Fatty alcohol forming acyl reductase (far) variants and methods of use

ABSTRACT

The present disclosure provides methods useful for producing fatty alcohol compositions from recombinant host cells. The disclosure further provides fatty acyl-CoA reductase (FAR) variant enzymes, polynucleotides encoding the FAR variant enzymes, and vectors and host cells comprising polynucleotides encoding the FAR variant enzymes.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. Provisional Application No. 61/577,756, filed Dec. 20, 2011; of U.S. Provisional Application No. 61/578,673, filed Dec. 21, 2011; of U.S. Provisional Application No. 61/636,044, filed Apr. 20, 2012; and of U.S. Provisional Application No. 61/674,053, filed Jul. 20, 2012; the entire content of each of which is incorporated herein by reference.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED AS AN ASCII FILE

The Sequence Listing written in file 90834-853678_ST25.TXT, created on Dec. 13, 2012, 121,968 bytes, machine format IBM-PC, MS-Windows operating system, is hereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates to fatty alcohol forming acyl reductases (FAR) variants, recombinant bacterial microorganisms incorporating polynucleotides encoding the FAR variants, and production of fatty alcohols by the engineered bacterial microorganisms.

BACKGROUND OF THE INVENTION

Crude petroleum has traditionally been used as a primary source for raw materials for producing numerous specialty chemicals. Specialty chemicals that can be produced from the petrochemical raw materials include fatty alcohols. These fatty alcohols have many industrial and commercial uses. For illustration, fatty alcohols are components of commercial waxes, lubricating oils, cosmetics, and solvents. Medium chain fatty alcohols such as C12 and C14 fatty alcohols act as surfactants and are used as ingredients in the personal care industry and in the manufacture of detergent.

Fatty alcohols can be obtained from crude petroleum. However, this process requires a significant amount of energy and involves the use of a non-renewable energy source. There is a need for improved methods for producing fatty alcohols, such as medium chain fatty alcohols.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the invention provides microorganisms engineered to produce a fatty alcohol composition. In some embodiments, the microorganism comprises a polynucleotide sequence encoding a variant fatty alcohol forming acyl-CoA reductase (FAR) polypeptide, wherein said variant FAR comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:2 and further comprising a substitution at one or more positions selected from positions 18, 65, 128, 134, 138, 177, 188, 224, 226, 405, 418, 433, 458, 487, 502, 508, 509, and 511, wherein the position is numbered with reference to SEQ ID NO:2, and wherein the microorganism produces a fatty alcohol composition comprising at least 20% of C12 and C₁₋₄ alcohols.

In some embodiments, the microorganism comprises a substitution selected from Q18I/L, R65G, N128C/H/L, N134D/K/R/S/Y, E138Q/R, N177D/E/L/R/T, P188D/E/I/R/S/W, K224R, L226M, P405A/C/F/G/L/V, Q418I/N/R/V, S433H/K/N/R/Y, S458E/N/Q, G487R/Y, L502A/Q/R/S/W, R508D/G/H/L/M, K509D/G/H/P/Q/R, and A511D/G/I/K/P/R/S/T. In some embodiments, the substitution is selected from Q181/L, R65G, N128H, N134S, E138Q, N177T, P188S, K224R, L226M, P405V, Q418V, S433K, S458Q, G487R, L502S, R508D, K509D, and A511T. In some embodiments, the microorganism further comprises a substitution at one or more positions selected from positions 4, 6, 7, 14, 62, 104, 108, 189, 220, 227, 316, 318, 355, 361, 365, 401, 410, 496, 507, and 510, wherein the position is numbered with reference to SEQ ID NO:2. In some embodiments, the substitution is selected from Q4/N/R/S/W/Y, Q6C/H/K/P/R/S/V/Y, Q7H/N, G14K/L/M/R/V, P62Q, V104I/M, R108E/G/H/Q, A189L/N, R220A/H, E227G, I316L, V318F/L/M, I355F/L/S/W, 1361C/F/L, M365N, G401A/C/L/S/TN, G410D/H/R, E496D/G, T507H/Q, and K510D/E/L/M/N/R/S. In some embodiments, the substitution is selected from Q4Y, Q6R, Q7N, G14L, P62Q, V104I, R108H, A189N, R220H, E227G, I316L, V318F, I355L, I361F, M365N, G401V, G410H, E496G, T507H, and K510E. In some embodiments, the microorganism comprises substitutions at two, three, four, five, six, seven, eight, nine, ten or more positions. In some embodiments, the microorganism comprises the amino acid sequence of any of SEQ ID NOs:6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or 28. In some embodiments, the microorganism comprises a functional fragment of a FAR variant sequence as described herein.

In some embodiments, the microorganism comprises a polynucleotide sequence encoding a variant fatty alcohol forming acyl-CoA reductase (FAR) polypeptide, wherein said variant FAR comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:2 or SEQ ID NO:4 and further comprising a substitution at one or more positions selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 23, 40, 43, 44, 45, 47, 49, 50, 52, 61, 62, 63, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 83, 84, 85, 86, 87, 88, 89, 90, 91, 93, 97, 98, 100, 101, 103, 104, 106, 107, 108. 111, 112, 113, 115, 116, 118, 120, 121, 122, 123, 126, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 140, 144, 145, 148, 150, 151, 153, 154, 155, 156, 157, 158, 160, 161, 162, 163, 164, 166, 167, 174, 176, 177, 178, 179, 180, 181, 182, 185, 186, 187, 188, 189, 190, 191, 192, 193, 195, 196, 197, 198, 200, 205, 206, 207, 208, 209, 211, 212, 215, 216, 218, 220, 221, 224, 225, 226, 227, 228, 231, 235, 236, 238, 239, 240, 241, 242, 244, 245, 246, 247, 253, 258, 263, 264, 266, 267, 268, 270, 273, 275, 277, 278, 280, 281, 283, 284, 285, 286, 288, 303, 306, 308, 310, 313, 316, 318, 331, 337, 338, 339, 341, 351, 352, 355, 359, 361, 362, 363, 365, 368, 370, 373, 374, 376, 377, 380, 382, 384, 387, 388, 389, 393, 396, 397, 398, 399, 400, 401, 402, 404, 405, 406, 308, 409, 410, 411, 412, 413, 414, 416, 417, 418, 419, 421, 424, 426, 429, 430, 432, 433, 436, 442, 443, 446, 452, 458, 463, 465, 466, 474, 478, 482, 487, 489, 490, 491, 494, 495, 496, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, or 512, wherein the position is numbered with reference to SEQ ID NO:2, and wherein the microorganism produces a fatty alcohol composition comprising at least 30% of C12 and C14 alcohols. In some embodiments, the substitution is selected from M1E/G/L/R/V/W, A2D/F/G/H/P/Q/R/S/T/W/Y, T3/I/L, Q4/N/R/S/W/Y, Q5M/N, Q6C/H/K/P/R/S/V/Y, Q7H/N, N8A/E/H/V, G9C/FN, A10T, S11D/G, A12D/R/S/T, S13G/L/V, G14K/L/M/R/V, V151, L16G/I/S, E17C/G/H/R, Q18I/L, R20K, H23R, K40R, R43H, T44A, V45A/S, D47E/N, G49E, G50A, H52Y, H61R, P62Q, A63D, R65G, E66D/F/S/Y, F68AN, L69E/I/M/Q, N70D/E/L/M/R/T, E71C/M/Q/S, 172L, A73G/H/K/L/M, S74K/L/P/T/W, S75C/E/H/N, S76E/F/I/L/R, V77I/P/T, F78M, E79D/1/L/Q/V, R80I/L, L81F/T, H83E, D84A, D85E, N86S, E87V, A88G/V, F89D/N/P/R, E90D/Q, T91A/R, L93D, V97I, H98P, 1100V, T101A, E103C/S/V, V104I/M, E106A/H, S107A, R108E/G/H/Q, L111I, T112G, P113I/Q, R115D, F116Y, A118K, A120C, G121T, Q122E/H/T, V123L, F126V, N128C/H/L, S129D, A130C/S, A131P/S, S132H, V133A/G, N134D/K/R/S/V, F135E, R136L, E137L, E138Q/R, D140Y, K144NE/R, 1145E/H, L148E/K/T, L150P, E151G/RN, V153F/I, A154G/R, A155G/M/R/T/W, L156M, A157Q/V, E158D/N, N160T, S161P/Y, A162K, M163L, A164V, 1166L/M, Q167H, N174A, K176G/I/M, N177D/E/L/R/T, S178F/L, G179D/S/W, Q180C/R, 1181D/E/L/V, T182G/I/K/R, V185G/I/P, 1186H, K187P, P188D/E/I/R/S/W, A189UN, G190I/K/L, E191V/W, S192A, I193C/L/V, R195F/H/I/N/W, S196D, T197F/P, D198S, Y200F, E205K, L206C, V207L/M, H208R, L209N/T/Y, Q211H/L/N/R, D212F, S215E/Y, D216G/Q, K218P/Q/R, R220A/H, Y221D/K, K224R, V225C/M, L226M, E227G, K228H, V231A, 1235E, R236I, A238G, N239C, N240Q/R/T, Y241F, G242E, S244A/P/R, D245H, T246A/P/V, Y247N, L253P/V, L258P, S263N, G264R, S266A/T, L267H, T268N, V270L, S273F, 1275V, S277A, A278C, E280I, E281S/Y, S283A/E/F/M/T, P284C/L/Q, G285D, W286Y, E288D/H/Q, E303G, S306T/W, F3081, G310LN, S313Q, I316L, V318F/L/M, S331V, S337G, G338E, S339P, Q341R, G351C, S352G, I355F/L/S/W, K359E, I361C/F/L, D362L, Y363H, M365N, A368S, T370A/I, A373W, A374Y, D376K/P/R, Q377H/K, Y380H/R, R382H/Q, T384S, F387I/L, V388L, A389I/M/L/V, K393A, D396G, V397L, V398Y, V399I, G400S, G401A/C/L/S/T/V, M402V, V404I/L, P405A/C/F/G/L/V, L406Y, 1408L, A409T/V/W/Y, G410D/H/R, K411R, A412V, M413L/R, R414K, A416L/V, G417V, Q418I/N/R/V, N419S, E421D/G/I/L/P/R/S/V, V424M, K426R/T, D429E/FUR, T430A/H/I, R432C/Q, S433H/K/N/R/Y, T436A, T442I, A443T, Y446F, S452E/G/N, S458E/N/Q, L463V, R465K, V466G/Q, Q474L/R, Q478E, C482R, G487R/Y, L489F, N490C/S, R491M, L494Y, K495C/S, E496D/G, K498G/N, L499R/S/V, Y500H/N, S501C/F/W, L502A/Q/R/S/W, R503C/K, A504D/E/S/T, A505E/G/K, D506G/L/M/R/W, T507H/Q, R508D/G/H/L/M, K509D/G/H/P/Q/R, K510D/E/L/M/N/R/S, A511D/G/I/K/P/R/S/T and/or A512M/R.

In some embodiments, the microorganism comprises a polynucleotide sequence encoding a FAR variant comprising an amino acid substitution set selected from the substitution sets listed in Table 1, Table 2, Table 4, Table 7, Table 8, Table 9, Table 10, or Table 11.

In some embodiments, the microorganism is a bacterial microorganism. In some embodiments, the microorganism is E. coli.

In some embodiments, the microorganism produces a fatty alcohol composition comprising at least 30% of C12 and C14 fatty alcohols. In some embodiments, the microorganism produces a fatty alcohol composition comprising at least 60% of C12 and C14 fatty alcohols. In some embodiments, the microorganism produces a fatty alcohol composition comprising at least 40% of C12 fatty alcohol. In some embodiments, the microorganism produces a fatty alcohol composition comprising from about 40% to about 75% of C12 fatty alcohol. In some embodiments, the microorganism produces a fatty alcohol composition comprising no more than about 30% of C14 fatty alcohol. In some embodiments, the microorganism produces a fatty alcohol composition comprising from about 20% to about 30% of C14 fatty alcohol.

In another aspect, the invention provides a fatty alcohol composition produced by a microorganism as described herein.

In another aspect, the invention relates to an isolated variant fatty alcohol forming acyl-CoA reductase (FAR) polypeptide. In some embodiments, the FAR variant polypeptide comprises an amino acid substitution set selected from the substitution sets listed in Table 1, Table 2, Table 4, Table 7, Table 8, Table 9, Table 10, or Table 11. In some embodiments, the polypeptide comprises the amino acid sequence of any of SEQ ID NOs:6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or 28.

In another aspect, the invention relates to a recombinant polynucleotide comprising a sequence encoding a variant FAR polypeptide encompassed by the invention. In some embodiments, the polynucleotide comprises the nucleotide sequence of any of SEQ ID NOs:5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27. In some embodiments, the polynucleotide is a codon optimized polynucleotide.

In another aspect, the invention relates to a vector comprising a polynucleotide sequence encoding a FAR variant polypeptide and optionally comprising one or more control sequences, such as a promoter capable of mediating expression of the polynucleotide encoding the FAR variant polypeptide in a host microorganism.

In another aspect, the invention relates to a host cell comprising a polynucleotide sequence encoding a FAR variant polypeptide.

In another aspect, a FAR variant protein, vectors and cells comprising a nucleic acid encoding the FAR variant protein, microorganisms engineered to express the FAR variant protein, and fatty alcohol products obtained from the microorganisms are provided, where the FAR variant protein has 100% identity to SEQ ID NO:2 except for the substitions present in any individual FAR variant selected from variant numbers 1-1046 in Table 1, Table 2, Table 4, Table 7, Table 8, Table 9, or Table 10. In other embodiments, the FAR variant protein has 100% identity to SEQ ID NO:2 except for (1) the substitions present in any individual FAR variant selected from variant numbers 1-1046 in Table 1, Table 2, Table 4, Table 7, Table 8, Table 9, or Table 10 and (2) one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve additional substitutions, e.g., 1-5, 2-6, 4-8, or 5-12 substitutions (which optionally are conservative substitutions).

In another aspect, a FAR variant protein, vectors and cells comprising a nucleic acid encoding the FAR variant protein, microorganisms engineered to express the FAR variant protein, and fatty alcohol products obtained from the microorganisms are provided, where the FAR variant protein has 100% identity to SEQ ID NO:4 except for the substitions present in any individual FAR variant selected from variant numbers 1047-1070 in Table 11. In other embodiments, the FAR variant protein has 100% identity to SEQ ID NO:4 except for (1) the substitions present in any individual FAR variant selected from variant numbers 1047-1070 in Table 11 and (2) one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve additional substitutions, e.g., 1-5, 2-6, 4-8, 5-12 substitutions (which optionally are conservative substitutions).

In another aspect, the invention relates to methods of producing a fatty alcohol composition comprising culturing a microorganism (e.g., E. coli) comprising a variant FAR polypeptide encompassed by the invention in a suitable culture medium in the presence of a carbon source under conditions in which fatty alcohols are produced. In some embodiments, at least 2 g/L (e.g., at least 2.5 g/L, at least 3 g/L, at least 3.5 g/L, at least 4 g/L, at least 4.5 g/L, at least 5 g/L, at least 10 g/L, at least 20 g/L, at least 30 g/L, at least 40 g/L, or at least 50 g/L) of recoverable fatty alcohols are produced. In some embodiments, the method further comprises recovering the fatty alcohol composition.

In another aspect, the invention relates to compositions and/or derivatives of the fatty alcohol compositions produced by the methods of the invention. In some embodiments, the fatty alcohol compositions produced by the methods encompassed by the invention, or a fraction thereof, are further reduced to yield an alkane composition. In some embodiments the fatty alcohol composition produced by the methods encompassed by the invention are esterified yielding fatty esters. In some embodiments, the fatty alcohol compositions produced by the methods encompassed by the invention, or a fraction thereof, are modified to produce fatty esters. In some embodiments, the composition is a detergent composition. In some embodiments, the composition is a personal care composition.

In another aspect, the invention provides methods of producing a detergent composition, the method comprising combining the fatty alcohols produced by the methods described herein, or a fraction thereof, with a detergent component selected from sodium carbonate, a complexation agent, zeolites, a protease, a lipase, amylase, carboxymethyl cellulose, optical brighteners, colorants and perfumes, thereby producing the detergent composition. In another aspect, the invention relates to detergent compositions produced by a method described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Relative fatty alcohol chain length distribution for FAR variants.

FIG. 2. Alignment of FAR proteins closely related to the Marinobacter algicola FAR (ZP_(—)01892457). Only the first ˜300 amino acids of the alignment are shown. The cofactor binding domain TGxxGxxG and the 2 putative YxxxK motifs are indicated. 1=Oceanobacter RED65; 2=Marinobacter aquaeolei; 3=Marine bacteria HP15; 4=Hahella KCTC 2396; 5=Marinobacter algicola.

FIG. 3. Alignment of FARs from plants, silk moth, mouse, and Marinobacter algicola. Only the active site motif (YxxxKxxxE) region is shown. 1=Arabidopsis CER4 (AAL49822); 2=Jojoba (AAD38039); 3=Arabidopsis (ABZ10954); 4=Arabidopsis (ABZ10951); 5=Arabidopsis (ABZ10952); 6=Arabidopsis (ABZ10953); 7=Wheat TAA1a (CAD67817); 8=Silk moth (NP_(—)001036967); 9=Mouse (AAH07178); 10=M. algicola (ZP_(—)01892457).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein and the laboratory procedures of cell culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry described below are those well-known and commonly employed in the art. It is noted that the indefinite articles “a” and “an” and the definite article “the” are used in the present application to mean one or more unless the context clearly dictates otherwise. Further, the term “or” is used in the present application to mean the disjunctive “or” and the conjunctive “and.”

The terms “fatty alcohol forming acyl-CoA reductase,” “fatty acyl reductase,” “FAR”, “FAR polypeptide,” and “FAR enzyme” are used interchangeably herein to refer to an enzyme that catalyzes the reduction of a fatty acyl-CoA, a fatty acyl-ACP, or other fatty acyl thioester complex to a fatty alcohol, in a reaction linked to the oxidation of NAD(P)H to NAD(P)⁺, as shown in the following Scheme 1:

wherein “R” represents a C7 to C23 saturated, unsaturated, linear, branched or cyclic hydrocarbon chain, and “R₁” represents CoA, ACP or other fatty acyl thioester substrates. CoA is a non-protein acyl carrier group factor (or moiety) involved in the synthesis and oxidation of fatty acids. “ACP” is a polypeptide or protein subunit of fatty acid synthase used in the synthesis of fatty acids. In some embodiments, a FAR catalyzes the reduction of a fatty acyl-CoA, a fatty acyl-ACP, or other fatty acyl thioester complex to a fatty aldehyde intermediate, which is reduced to a fatty alcohol by a second oxidoreductase enzyme.

“Fatty aldehyde” as used herein refers to a saturated or unsaturated aliphatic aldehyde.

The term “fatty acid” as used herein refers to a compound having the formula RCO—OH, or a salt thereof, wherein “R” is as defined above. In some embodiments, the fatty acid salt is a potassium salt, a sodium salt, or an ammonium salt. Saturated or unsaturated fatty acids can be described as “Ca:b”, wherein “a” is an integer that represents the total number of carbon atoms and “b” is an integer that refers to the number of double bonds in the carbon chain.

The term “fatty alcohol” as used herein refers to an aliphatic alcohol of the formula R—OH, where R is as defined above. Saturated or unsaturated fatty alcohols can also be described using the nomenclature “Ca:b” or, alternatively “Ca:b-OH”, wherein “a” is an integer that represents the total number of carbon atoms in the fatty alcohol and “b” is an integer that refers to the number of double bonds in the carbon chain. In some embodiments, a fatty alcohol produced according to the methods disclosed herein is a C8-C24 saturated or unsaturated fatty alcohol (i.e., a C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, or C24 fatty alcohol). In some embodiments, one or more of the following fatty alcohols are present: 1-decanol, 1-dodecanol, 1-tetradecanol, 1-hexadecanol and 1-octadecanol.

Unsaturated fatty acids or fatty alcohols can be referred to as “cis Δ^(x)” or “trans Δ^(x)”, wherein “cis” and “trans” refer to the carbon chain configuration around the double bond and “x” indicates the number of the first carbon of the double bond, wherein carbon 1 is the carboxylic acid carbon of the fatty acid or the carbon bound to the —OH group of the fatty alcohol.

A “fatty alcohol composition” refers to fatty alcohols produced from a recombinant microorganism. A fatty alcohol composition may comprise a plurality (e.g., combination) of fatty alcohols, such as but not limited to fatty alcohols having a carbon chain length of C12, C14, C16, and C18. In one embodiment, a fatty alcohol composition comprises predominantly fatty alcohols having a specific carbon chain length (such as but not limited to C12 or C14 fatty alcohols). The fatty alcohol composition may comprise saturated, unsaturated, and/or branched fatty alcohols. As used herein, the phrase “C12 to C14 fatty alcohols” means C12 and C14 fatty alcohols; similarly, the phrases “C12 to C16 fatty alcohols”; “C14 to C16 fatty alcohols”; and “C12 to C18 fatty alcohols” mean C12, C14, and C16 fatty alcohols; C14 and C16 fatty alcohols; and C12, C14, C16, and C18 fatty alcohols, respectively.

The terms “fatty acyl-thioester” and “fatty acyl-thioester complex” refer to a compound of formula (I) in Scheme 1, in which a fatty acyl moiety is covalently linked via a thioester linkage to a carrier moiety. Fatty acyl-thioesters are substrates for the improved FAR polypeptides described herein.

The term “fatty acyl-CoA” refers to a compound of formula (I) in Scheme 1, wherein R₁ is Coenzyme A (“CoA”).

The term “fatty acyl-ACP” refers to a compound of formula (I) in Scheme 1, wherein R₁ is acyl carrier protein (“ACP”).

The term “fatty acid synthase” or “FAS” refers to an enzyme or enzyme complex that catalyzes the conversion of acetyl-CoA and malonyl-CoA to fatty acyl-ACP as set forth in the following Scheme 2:

wherein ACP is a protein which comprises a covalently attached phosphopantetheine moiety. In certain embodiments, the FAS is composed of more than one distinct enzymatic activity. In various embodiments, the distinct enzymatic activities reside in separate polypeptides. In some embodiments, the separate polypeptides form one or more protein complexes.

The term “acyl-ACP thioesterase (TE)” refers to an enzyme that catalyzes the cleavage of acyl-ACP to form a fatty acid, as shown in the following Scheme 3, wherein R has the same meaning as set forth above:

The terms “fatty acyl-CoA synthetase,” “acyl-CoA synthetase,” and “FACS” are used interchangeably herein to refer to an enzyme that catalyzes the formation of a covalent complex between the acyl portion of the fatty acid and CoA as shown in the following Scheme 4, wherein R has the same meaning as set forth above:

The term “acetyl-CoA carboxylase” or “ACC” refers to an enzyme that catalyzes the conversion of acetyl-CoA to malonyl-CoA as shown in the following Scheme 5:

The term “acyl-CoA dehydrogenase” or “ACD” refers an enzyme that catalyzes the introduction of a trans double-bond between C2 and C3 of an acyl-CoA thioester substrate as shown in the following Scheme 6:

“Conversion” refers to the enzymatic conversion of the substrate to the corresponding product.

“Naturally-occurring” or “wild-type” refers to the form found in nature. For example, a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a source in nature and which has not been intentionally modified by human manipulation. A wild-type organism or cell refers to an organism or cell that has not been intentionally modified by human manipulation.

The term “wild-type fatty alcohol forming acyl-CoA reductase” or “wild-type FAR,” as used herein, refers to a naturally-occurring FAR polypeptide. In some embodiments, a wild-type FAR is produced by a gammaproteobacteria, including but not limited to strains of Marinobacter, Oceanobacter, and Hahella. Naturally occurring FAR polypeptides are described, for example, in US patent publication 2011/0000125, incorporated by reference herein. In some embodiments, a wild-type FAR is a naturally-occurring FAR polypeptide that is produced by the Marinobacter algicola strain DG893 (SEQ ID NO:2). In some embodiments, a wild-type FAR is a naturally-occurring FAR polypeptide that is produced by the Marinobacter aquaeolei strain VT8 (SEQ ID NO:4). FARs that are not produced in nature can be denoted “recombinant” FARs, whether prepared using recombinant techniques or by chemical synthesis.

The term “FAR variant” refers to a FAR polypeptide having substitutions at one or more positions relative to a wild-type FAR polypeptide and to functional (or “biologically active”) fragments thereof. In one embodiment, “FAR variants” comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:2 or a functional fragment thereof. In another embodiment, “FAR variants” comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 4, or a functional fragment thereof.

In the context of a FAR polypeptide, “variant” refers to a FAR polypeptide or polynucleotide comprising one or more modifications relative to a wild-type FAR polypeptide such as wild-type FAR from Marinobacter species. In the context of a polynucleotide encoding a FAR polypeptide, “variant” refers to a polynucleotide encoding a FAR variant.

The terms “modifications” and “mutations,” when used in the context of substitutions, deletions, insertions and the like with respect to polynucleotides and polypeptides, are used interchangeably herein and refer to changes that are introduced by genetic manipulation to create variants from a wild-type sequence.

“Deletion” refers to modification to a polypeptide by removal of one or more amino acids relative to a reference polypeptide. Deletions can comprise removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids making up the reference polypeptide while retaining enzymatic activity or having improved improperties (e.g., improved enzymatic activity) relative to the reference polypeptide. Deletions can be directed to the internal portions and/or terminal portions of the polypeptide. In one embodiment, the deletion comprises the removal of a continuous amino acid segment. In another embodiment, the deletion comprises the removal of two or more amino acids or amino acid segments that are discontinuous (i.e., two or more amino acids or amino acid segments separated by one or more amino acid residues that are not removed from the reference polypeptide). The term “deletion” is also used to a DNA modification in which or more nucleotides or nucleotide base-pairs have been removed, as compared to the corresponding reference, parental, or wild-type DNA.

“Insertion” refers to modification to a polypeptide by addition of one or more amino acids to the reference polypeptide. In some embodiments, the modification comprises insertions of one or more amino acids to the naturally occurring polypeptide as well as insertions of one or more amino acids to other modified polypeptides. Insertions can be in the internal portions, or in the carboxy or amino terminus. “Insertions,” as used herein, includes fusion proteins as is known in the art.

A FAR polypeptide (i.e., a FAR variant) is “derived from” a wild-type FAR polypeptide sequence by introducing modifications (e.g., amino acid substitutions) into a wild-type sequence (e.g., the wild-type FAR polypeptide of SEQ ID NO:2 or SEQ ID NO:4) using in vitro mutagenesis or molecular evolution methods known in the art. Typically, the polypeptide sequence of a FAR variant will be at least 70% (alternatively, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the wild-type sequence.

“Percentage of sequence identity,” “percent identity” and “percentage homology” are used interchangeably herein to refer to comparisons among polynucleotides and polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which may also contain gaps to optimize the alignment) for alignment of the two sequences. The percentage may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (including positions where one of the sequences has a gap(s)) and multiplying the result by 100 to yield the percentage of sequence identity. For example, a polypeptide with an amino acid sequence matching SEQ ID NO:2 at 491 positions, with one gap, would have 491/512=95.9% identity to SEQ ID NO:2. Similarly, a FAR variant that has 475 residues (i.e., less than full-length) and matches SEQ ID NO:2 at 460 positions would have 460/475=96.8% identity. Those of skill in the art appreciate that there are many established algorithms available to align two sequences and that different methods may give slightly different results.

Alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection (see generally, Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)). The Clustral (Chema R., Sugawara H., Koike T., Lopez R., Gibson T. J., Higgins D. G., Thompson J. D., (2003) Multiple sequence alignment with the Clustral series of programs, Nucleic Acids Res., 31, 3497-3500.) and T-Coffee (T-COFFEE: A novel method for multiple sequence alignments. Notredame, Higgins, Hering a, JMB 302 (205-217) 2000 software packages may also be used to align sequences.

Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1990, J. Mol. Biol. 215: 403-410 and Altschul et al., 1977, Nucleic Acids Res. 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as, the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=−4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA 89:10915). Exemplary determination of sequence alignment and % sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison Wis.), using default parameters provided.

“Reference sequence” refers to a defined sequence used as a basis for a sequence comparison. In some cases, a “reference sequence” refers to the sequence of a wild-type FAR (e.g., SEQ ID NO:2 or 4) or the sequence of a specified FAR variant (e.g., SEQ ID NO:6, 8, or 10). A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotide or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, at least 100 residues in length or the full length of the nucleic acid or polypeptide. Since two polynucleotides or polypeptides may each (1) comprise a sequence (i.e., a portion of the complete sequence) that is similar between the two polynucleotides or polypeptides, and (2) may further comprise a sequence that is divergent between the two polynucleotides or polypeptides, sequence comparisons between two (or more) polynucleotides or polypeptide are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity.

“Comparison window” refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acids residues wherein a sequence may be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids and wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The comparison window can be longer than 20 contiguous residues, and includes, optionally 30, 40, 50, 100, or longer windows.

Nucleic acids “hybridize” when they associate, typically in solution. Nucleic acids hybridize due to a variety of well-characterized physico-chemical forces, such as hydrogen bonding, solvent exclusion, base stacking and the like. As used herein, the term “stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments, such as Southern and Northern hybridizations, are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) “Laboratory Techniques in biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes,” Part I, Chapter 2 (Elsevier, New York), which is incorporated herein by reference. For polynucleotides of at least 100 nucleotides in length, low to very high stringency conditions are defined as follows: prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and either 25% formamide for low stringencies, 35% formamide for medium and medium-high stringencies, or 50% formamide for high and very high stringencies, following standard Southern blotting procedures. For polynucleotides of at least 100 nucleotides in length, the carrier material is finally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at least at 50° C. (low stringency), at least at 55° C. (medium stringency), at least at 60° C. (medium-high stringency), at least at 65° C. (high stringency), and at least at 70° C. (very high stringency).

“Codon optimized” refers to changes in the codons of the polynucleotide encoding a protein to those preferentially used in a particular organism such that the encoded protein is efficiently expressed in the organism. Although the genetic code is degenerate in that most amino acids are represented by several codons, called “synonyms” or “synonymous” codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome. In some embodiments, the polynucleotides encoding enzymes may be codon optimized for optimal production from the host organism selected for expression. For example, in some embodiments, a polynucleotide encoding a FAR variant as described herein (e.g., comprising one or more substitution sets listed in Table 1, Table 2, Table 4, Table 7, Table 8, Table 9, Table 10, or Table 11) is codon optimized for expression in bacteria, e.g., E. coli.

“Preferred, optimal, high codon usage bias codons” refers interchangeably to codons that are used at higher frequency in the protein coding regions than other codons that code for the same amino acid. The preferred codons may be determined in relation to codon usage in a single gene, a set of genes of common function or origin, highly expressed genes, the codon frequency in the aggregate protein coding regions of the whole organism, codon frequency in the aggregate protein coding regions of related organisms, or combinations thereof. Codons whose frequency increases with the level of gene expression are typically optimal codons for expression. A variety of methods are known for determining the codon frequency (e.g., codon usage, relative synonymous codon usage) and codon preference in specific organisms, including multivariate analysis, for example, using cluster analysis or correspondence analysis, and the effective number of codons used in a gene (See GCG Codon Preference, Genetics Computer Group Wisconsin Package; CodonW, John Peden, University of Nottingham; McInerney, J. O, 1998, Bioinformatics 14:372-73; Stenico et al., 1994, Nucleic Acids Res. 222437-46; Wright, F., 1990, Gene 87:23-29). Codon usage tables are available for a growing list of organisms (see for example, Wada et al., 1992, Nucleic Acids Res. 20:2111-2118; Nakamura et al., 2000, Nucleic Acids Res. 28:292; Henaut and Danchin, “Escherichia coli and Salmonella,” 1996, Neidhardt, et al. Eds., ASM Press, Washington D.C., p. 2047-2066). The data source for obtaining codon usage may rely on any available nucleotide sequence capable of coding for a protein. These data sets include nucleic acid sequences actually known to encode expressed proteins (e.g., complete protein coding sequences-CDS), expressed sequence tags (ESTs), or predicted coding regions of genomic sequences (see for example, Mount, D., Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Uberbacher, E. C., 1996, Methods Enzymol. 266:259-281; Tiwari et al., 1997, Comput. Appl. Biosci. 13:263-270).

In describing the variants and amino acid substitutions of the present invention, the nomenclature described below is used. In all cases the accepted IUPAC single letter or triple letter amino acid abbreviations are employed. IUPAC single letter amino acid abbreviations are as follows: alanine (A); cysteine (C); aspartic acid (D); glutamic acid (E); phenylalanine (F); glycine (G); histidine (H); isoleucine (I); lysine (K); leucine (L); methionine (M); asparagine (N); proline (P); glutamine (Q); arginine (R); serine (S); threonine (T); valine (V); tryptophan (W); and tyrosine (Y). For amino acid substitutions relative to a specified sequence, the following nomenclature is used: [Original amino acid, position, substituted amino acid]. As a non-limiting example, for a variant polypeptide described with reference to SEQ ID NO:2, “A2V” indicates that in the variant polypeptide, the alanine at position 2 of the reference sequence is replaced by valine, with amino acid position being determined by optimal alignment of the variant sequence with SEQ ID NO:2. Similarly, “A512K/S/T” describes three variants: a variant in which the alanine at position 512 of the reference sequence is replaced by lysine, a variant in which the alanine at position 512 of the reference sequence is replaced by serine, and a variant in which the alanine at position 512 of the reference sequence is replaced by threonine. In some embodiments, an amino acid (or base) may be called “X,” by which is meant any amino acid (or base). For example, X2D/F/G/H/I/P/N/Q/T/V/W can refer to a substitution in a FAR homolog in which the residue (X) at the position in the homolog corresponding to position 2 of a specified sequence (e.g., SEQ ID NO:2) is substituted so that the residue at position 2 is any of D, F, G, H, I, P, N, Q, T, V, and W.

The term “amino acid substitution set” or “substitution set” refers to a group of amino acid substitutions. A protein characterized as comprising a particular “substitution set” comprises the substitutions of the substitution set relative to a reference amino acid sequence. A substitution set can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions. Exemplary substitution sets are provided in Tables 1, 2, 4, and 7-11. For example, the substitution set for Variant 26 (Table 1) consists of the amino acid substitutions N134S, E138Q, P188S, and A511T. Examples of polypeptides comprising this substitution set include, without limitation, Variant 26 and each of Variants 27-33. In some embodiments, a substitution set is described relative to a reference amino acid sequence that is a FAR variant and comprises the substitutions of the reference amino acid sequence. For example, the substitution set for Variant 144 (Table 2) consists of the amino acid substitutions G65R, S266A, R382H, A389M, and G401V relative to SEQ ID NO:10. SEQ ID NO:10 is FAR Variant 129, which has the substitution set Q181, R65G, N128H, N134S, E138Q, N177T, P188S, K224R, L226M, P405V, Q418V, S433K, S458Q, G487R, L502S, R508D, K509D, and A511T relative to SEQ ID NO:2. Accordingly, Variant 144 also comprises the amino acid substitutions Q181, R65G, N128H, N134S, E138Q, N177T, P188S, K224R, L226M, P405V, Q418V, S433K, S458Q, G487R, L502S, R508D, K509D, and A511T relative to SEQ ID NO:2.

A “conservative substitution,” as used with reference to amino acids, refers to the substitution of an amino acid with a chemically similar amino acid. Amino acid substitutions which often preserve the structural and/or functional properties of the polypeptide in which the substitution is made are known in the art and are described, for example, by H. Neurath and R. L. Hill, 1979, in “The Proteins,” Academic Press, New York. The most commonly occurring exchanges are isoleucine/valine, tyrosine/phenylalanine, aspartic acid/glutamic acid, lysine/arginine, methionine/leucine, aspartic acid/asparagine, glutamic acid/glutamine, leucine/isoleucine, methionine/isoleucine, threonine/serine, tryptophan/phenylalanine, tyrosine/histidine, tyrosine/tryptophan, glutamine/arginine, histidine/asparagine, histidine/glutamine, lysine/asparagine, lysine/glutamine, lysine/glutamic acid, phenylalanine/leucine, phenylalanine/methionine, serine/alanine, serine/asparagine, valine/leucine, and valine/methionine.

In some embodiments, conservatively substituted variations of a polypeptide (e.g., a FAR polypeptide) includes substitutions of one or more amino acids of the polypeptide with a conservatively selected amino acid of the same conservative substitution group. In some embodiments less than 10%, less than 5%, less than 2% and sometimes less than 1% of the amino acids of the polypeptide are replaced. In some embodiments, there may be at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, or at least 40 conservative substitutions in a polypeptide. In some embodiments, there is no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 15, no more than 20, no more than 25, no more than 30, no more than 35, or no more than 40 conservative substitutions in a polypeptide. The addition of sequences which do not alter the encoded activity of a polynucleotide (e.g., a FAR polynucleotide), such as the addition of a non-functional or non-coding sequence, is considered a conservative variation of the polynucleotide.

“Functional fragment” (or “biologically active fragment”), as used herein, refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion and/or internal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the sequence to which it is being compared (e.g., a full-length FAR variant of the invention) and that retains substantially all of the activity of the full-length polypeptide. Functional fragments of variant FARs can comprise up to 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the corresponding full-length variant FAR enzyme.

An “endogenous” polynucleotide, gene, promoter or polypeptide refers to any polynucleotide, gene, promoter or polypeptide that originates in a particular host cell. A polynucleotide, gene, promoter or polypeptide is not endogenous to a host cell if it has been removed from the host cell, subjected to laboratory manipulation, and then reintroduced into a host cell.

A “heterologous” polynucleotide, gene, promoter or polypeptide refers to any polynucleotide, gene, promoter or polypeptide that is introduced into a host cell that is not normally present in that cell, and includes any polynucleotide, gene, promoter or polypeptide that is removed from the host cell and then reintroduced into the host cell.

“Recombinant host cell,” “engineered host cell,” “recombinant microorganism,” and “engineered microorganism” are used interchangeably herein and refer to a microorganism (e.g., a bacteria, yeast, filamentous fungi, or algae) into which has been introduced a heterologous polynucleotide, gene, promoter, e.g., an expression vector, or to a microorganism (e.g., a bacteria, yeast, filamentous fungi, or algae) having a heterologous polynucleotide or gene integrated into the genome.

“Control sequence” is defined herein to include all components, which are necessary or advantageous for the expression of a polypeptide of the present disclosure. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a polypeptide.

“Operably linked” and “operably associated” are defined herein as a configuration in which a control sequence is appropriately placed at a position relative to the coding sequence of the DNA sequence such that the control sequence directs the expression of a polynucleotide and/or polypeptide.

“Promoter sequence” is a nucleic acid sequence that is recognized by a host cell for expression of the coding region. The control sequence may comprise an appropriate promoter sequence. The promoter sequence contains transcriptional control sequences, which mediate the expression of the polypeptide. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either endogenous or heterologous to the host cell.

The term “expression” includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

The terms “transform” or “transformation,” as used in reference to a cell, means a cell has a non-native nucleic acid sequence integrated into its genome or as an episome (e.g., plasmid) that is maintained through multiple generations.

The term “culturing” refers to growing a population of microbial cells under suitable conditions in a liquid or solid medium. In particular embodiments, culturing refers to the fermentative bioconversion of a substrate to an end product.

The term “recoverable,” as used in reference to producing a composition (e.g., fatty alcohols) by a method of the present invention, refers to the amount of composition which can be isolated from the reaction mixture yielding the composition according to methods known in the art.

II. Introduction

The present invention relates to, among other things, variant FAR enzymes with improved properties, polynucleotides encoding the variant FAR enzymes, recombinant microorganisms comprising a nucleic acid encoding a FAR variant, microorganisms capable of expressing the FAR variants, processes for producing fatty alcohols and other compositions derived therefrom using the FAR variants and the resultant compositions.

Wild-type FAR polypeptides have been described. See, e.g., WO 2011/008535 (published 20 Jan. 2011), incorporated by reference herein for all purposes. Certain FAR enzymes from gammaproteobacteria (e.g., strains of Marinobacter and Oceanobacter or taxonomic equivalents thereof) are capable of generating high yields of fatty alcohols when genes encoding these enzymes are expressed in heterologous cells. For example, the wild-type FAR enzyme of Marinobacter species algicola (strain DG893) is capable of producing a significantly increased yield of total fatty alcohol as compared to FAR enzymes from B. mori, when expressed in an E. coli host. The invention provides FAR variants with improved properties as compared to their wild-type counterparts.

SEQ ID NO:2 is the amino acid sequence of the wild-type FAR from Marinobacter algicola (strain DG893) and is described in WO 2011/008535. SEQ ID NO:1 is the wild-type polynucleotide sequence encoding the wild-type FAR protein from Marinobacter algicola strain DG893 (SEQ ID NO:2). SEQ ID NO:4 is the amino acid sequence of wild-type FAR from Marinobacter aquaeolei VT8, which is also described in WO 2011/008535. SEQ ID NO:3 is a polynucleotide sequence encoding the wild-type FAR protein from Marinobacter aquaeolei VT8 (SEQ ID NO:4) that is codon optimized for expression. Amino acid sequence identity between SEQ ID NO:2 and SEQ ID NO:4 is about 78%. See WO 2012/006114.

In one aspect, the invention relates to improved FAR polypeptides. In another aspect, it can be seen that substitutions introduced at numerous different amino acid (also referred to herein as “residue”) positions within a wild-type FAR (e.g., a FAR of SEQ ID NO:2 or SEQ ID NO:4) yield FAR variant polypeptides capable of catalyzing increased production of shorter chain (e.g., C12 to C14) fatty alcohols as compared to the wild-type FAR, as shown for example in Table 1, Table 2, Table 4, Table 7, Table 8, Table 9, Table 10, or Table 11. In a related aspect, the invention relates to polynucleotides that encode a FAR variant polypeptide capable of catalyzing increased production of C12 and C14 fatty alcohols as compared to the wild-type FAR. In a related aspect, the invention relates to microorganisms (e.g., bacterial microorganisms such as E. coli) comprising a polynucleotide sequence encoding a FAR variant polypeptide, wherein the microorganism produces a fatty alcohol composition having a fatty alcohol profile which comprises a higher percentage of C12 and C14 fatty alcohols as compared to the fatty alcohol profile of a fatty alcohol composition produced by a microorganism expressing a wild-type FAR.

Section IV (“FAR Variants”) and Section XI (“Examples”), below, describe exemplary FAR variants with improved properties. One such improved property, discussed in Section III (“Improved Properties of FAR Variants”) and Section XI, is that a cell expressing the FAR variant produces a more desired profile of fatty alcohols than a cell expressing a wild-type FAR. Sections V (“Polynucleotides and Expression Systems for Expressing FAR Variants”) and VI (“Host Cells Comprising FAR Variants”) describe polynucleotides and vectors, and cell systems, respectively, used to express FAR variant proteins in cells and to produce fatty alcohols. Section VII (“Methods of Producing Fatty Alcohols”) describes methods for producing fatty alcohols using recombinant cells and recovering the produced fatty alcohols. Section III (and various other sections) describes the characteristics of fatty alcohols produced according to the methods of the invention, including fatty alcohol production levels and fatty alcohol profiles. Section IX describes “Exemplary Compositions Containing Fatty Alcohols and Fatty Alcohol Derivatives.” Section X describes methods for producing FAR proteins.

III. Improved Properties of Far Variants

In one aspect, the invention provides FAR variants having improved properties over a wild-type FAR enzyme (e.g., SEQ ID NO:2 or 4) or over a reference sequence (e.g., SEQ ID NO:6, 8, 10, 12, or 14). For example, a host cell or microorganism expressing a FAR variant of the invention may have the improved property of increased fatty alcohol production compared to a cell expressing a wild-type FAR (also referred to herein as a “control cell”) and/or the fatty alcohols produced may have a different fatty alcohol profile than the fatty alcohol profile produced by the control cell. In some embodiments, a cell expressing a FAR variant produces (i.e., yields) an increased amount of fatty alcohols as compared to a wild-type FAR (e.g., SEQ ID NO:2 or 4) or a reference FAR variant (e.g., SEQ ID NO:6, 8, 10, 12, or 14). In some embodiments, a cell expressing a FAR variant has a fatty alcohol profile that comprises a higher percentage of shorter chain fatty alcohols (e.g., C12 and C14) as compared to a control cell expressing a reference FAR. In some embodiments, the fatty alcohol profile produced by a cell expressing a FAR variant is characterized by an increased amount of C12 fatty alcohol (e.g., C12:0 (1-dodecanol) and/or C12:1 (cis Δ⁵-dodecenol)), an increased amount of C14 fatty alcohol (e.g., C14:0 (1-tetradecanol) and/or C14:1 (cis Δ⁷-1-tetradecanol)), an increased amount of C12 and C14 fatty alcohols (e.g., C12:0 (1-dodecanol), C12:1 (cis Δ⁵-dodecenol), C14:0 (1-tetradecanol), and/or C14:1 (cis Δ⁷-1-tetradecanol)), a decreased amount of C18 fatty alcohol (e.g., C18:0 (1-octadecanol) or C18:1 (cis Δ¹¹-1-octadecenol), or combinations of these, as compared to a wild-type FAR (e.g., SEQ ID NO:2 or SEQ ID NO:4) or a reference FAR (e.g., any of SEQ ID NOs:6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or 28). As used herein, “increased production of shorter chain fatty alcohols” with a FAR variant refers to an increased amount of shorter chain fatty alcohols (e.g., C12 and C14 fatty alcohols) as compared to a reference FAR (e.g., a wild-type FAR), and/or an increased proportion of shorter chain fatty alcohols (e.g., C12 and C14 fatty alcohols) in a fatty alcohol composition produced with the FAR variant as compared to a reference FAR (e.g., a wild-type FAR).

Other improved properties of FAR variants include, but are not limited to, increased total fatty alcohol production; increased level of carbon chain saturation; increased fatty alcohol production at a specified culture pH (e.g., pH 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8) or over a broader pH range (e.g., pH 3.5-7); increased production of fatty alcohols at a specified culture temperature (e.g., about 28° C., about 30° C., about 35° C., about 37° C., or about 40° C.), or over a broader temperature range (e.g., 30° C.-42° C.).

Fatty Alcohol Production

In some embodiments, the engineered microorganisms according to the invention, such as E. coli, comprising a FAR variant as described herein, are capable of producing at least about 1.5-fold (e.g., at least about 2.0-fold, at least about 3.0-fold, at least about 4.0-fold, and at least about 5.0-fold) more fatty alcohols than a wild-type FAR corresponding to SEQ ID NO:2 or SEQ ID NO:4 when assayed under the same conditions. In some embodiments, the engineered microorganisms according to the invention, such as E. coli, comprising a FAR variant as described herein, are capable of producing at least about 1.5-fold more fatty alcohols than a reference FAR variant (e.g., a FAR variant having the amino acid sequence of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:14) when assayed under the same conditions. In some embodiments, the engineered microorganisms according to the invention, such as E. coli, comprising a FAR variant as described herein, are capable of producing a fatty alcohol profile having one or more of an increased amount of C12:0 (1-dodecanol), an increased amount of C12:1 (cis Δ⁵-dodecenol), an increased amount of C14:0 (1-tetradecanol), an increased amount of C14:1 (cis Δ⁷-1-tetradecanol), an increased amount of C16:0 (1-hexadecanol), an increased amount of C16:1 (cis Δ⁹-1-hexadecenol), a decreased amount of C18:0 (1-octadecanol), or a decreased amount of C18:1 (cis Δ¹¹-1-octadecenol) as compared to a wild-type FAR or reference FAR variant when assayed under the same conditions.

In some embodiments, a FAR variant polypeptide of the invention is capable of producing more fatty alcohol than a wild-type polypeptide corresponding to SEQ ID NO:2 or SEQ ID NO:4 when total fatty alcohol production is determined. “Total fatty alcohol,” as used herein, refers to the intracellular and secreted amount of fatty alcohol. In some embodiments, a FAR variant polypeptide of the invention is capable of producing more fatty alcohol than a wild-type polypeptide corresponding to SEQ ID NO:2 or SEQ ID NO:4 when secreted fatty alcohol production is determined. “Secreted fatty alcohol,” as used herein, refers to the extracellular fatty alcohol.

Fatty alcohol content can be measured using art known methods, such as methods described herein below. In some embodiments, an engineered microorganism of the invention comprising a FAR variant polypeptide as described herein is capable of producing more fatty alcohol than a wild-type polypeptide corresponding to SEQ ID NO:2 or SEQ ID NO:4 or a reference FAR variant (e.g., a FAR variant corresponding to SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:14) when fatty alcohol production is determined by gas chromatography. In certain embodiments, total fatty alcohol production is determined by measuring production of representative fatty alcohols such as C12:0 (1-dodecanol), C12:1 (cis Δ⁵-dodecenol), C14:0 (1-tetradecanol), C14:1 (cis Δ⁷-1-tetradecanol), C16:0 (1-hexadecanol), C16:1 (cis Δ⁹-1-hexadecenol), C18:0 (1-octadecanol), and/or C18:1 (cis Δ¹¹-1-octadecenol).

In some embodiments, the fatty alcohol compositions that are produced comprise saturated fatty alcohols. In some embodiments, the fatty alcohol compositions comprise unsaturated fatty alcohols. In some embodiments, the unsaturated fatty alcohols are monounsaturated fatty alcohols. In some embodiments, the fatty alcohol compositions comprise both saturated and unsaturated fatty alcohols, and the amount of unsaturated fatty alcohols is less than about 30%, such as less than about 20%, such as less than about 10%, such as less than about 5%, such as less than about 1% of the fatty alcohols present in the composition. In some embodiments, C12 to C18 fatty alcohols comprise at least about 85%, such as at least about 90%, such as at least about 92%, such as at least about 95%, such as at least about 97%, such as at least about 99% of the produced fatty alcohols. In certain embodiments, C12 to C16 fatty alcohols comprise about 80%, such as at least about 85%, such as at least about 90%, such as at least about 92%, such as at least about 95%, such as at least about 97%, such as at least about 99% by weight of the produced fatty alcohols. In certain embodiments, C12 to C14 fatty alcohols (e.g., C12 and C14 fatty alcohols) comprise at least about 20%, such as at least about 25%, such as at least about 30%, such as at least about 35%, such as at least about 40%, such as at least about 45%, such as at least about 50%, such as at least about 55%, such as at least about 60%, such as at least about 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90% of the produced fatty alcohols.

In some embodiments, C12 to C16 fatty alcohols comprise at least about 80%, at least about 85%, at least about 90%, or at least about 95% by weight of the total isolated fatty alcohols. In certain embodiments, C12 to C14 fatty alcohols (e.g., C12 and C14 fatty alcohols) comprise at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, or at least about 85% by weight of the total isolated fatty alcohols. In some embodiments, the fatty alcohol compositions that are recovered (e.g., the C12 to C16 fatty alcohols or the C12 to C14 fatty alcohols) comprise saturated fatty alcohols. In some embodiments, the fatty alcohol compositions that are recovered (e.g., the C12 to C16 fatty alcohols or the C12 to C14 fatty alcohols) comprise a mixture of saturated and unsaturated fatty alcohols.

Fatty Alcohol Profiles

In some embodiments, the engineered microorganisms of the invention comprising a FAR variant polypeptide as described herein produce fatty alcohol profiles that differ from the fatty alcohol profiles produced by wild-type FAR. A “fatty alcohol profile” refers to the chain length distribution in a composition containing fatty alcohols, or the chain length distribution of fatty alcohols produced by a cell. In some embodiments, the fatty alcohol profile contains saturated fatty alcohols, unsaturated fatty alcohols, or a mixture of saturated and unsaturated fatty alcohols. The degree of unsaturation and position within a chain can also vary. For example, a fatty alcohol may have 1, 2, 3, or more double bonds. Additionally, two fatty alcohols with the same chain length may each have a single double bond but at different positions. In some embodiments, the relative proportions of C12:0 (1-dodecanol), C12:1 (cis Δ⁵-dodecenol), C14:0 (1-tetradecanol), C14:1 (cis Δ⁷-1-tetradecanol), C16:0 (1-hexadecanol), C16:1 (cis Δ⁹-1-hexadecenol), C18:0 (1-octadecanol), or C18:1 (cis Δ¹¹-1-octadecenol) are measured to determine fatty alcohol profile. Fatty alcohol profiles can be measured using art known methods, such as methods described hereinbelow.

In some embodiments, the FAR variant has at least about 70% (or at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%) sequence identity to SEQ ID NO:2 or SEQ ID NO:4 and comprises one or more amino acid substitutions as described herein (e.g., one or more amino acid substitutions or amino acid substitution sets listed in Table 1, Table 2, Table 6, Table 7, Table 8, Table 9, Table 10, or Table 11), wherein a cell or microorganism in which the FAR variant is expressed produces a fatty alcohol profile that differs from the fatty alcohol profile that is produced by a corresponding cell of the same type expressing the wild-type FAR or reference FAR from which the FAR variant derived when cultured under the same conditions. For example, a cell (e.g., E. coli) expressing the FAR variant may produce a higher percentage of a particular fatty alcohol (e.g., a higher percentage of C12, C14, and/or C16) than the cell expressing the wild-type FAR; or produce a lower percentage of a particular fatty alcohol (e.g., a lower percentage of C18) than the cell expressing the wild-type; or produces a higher percentage of a range of fatty alcohols (such as a higher percentage of C12 to C16 or C12 to C14 fatty alcohols) than the cell expressing the wild-type. Generally, the fatty alcohol profiles produced by a host cell (e.g., E. coli) expressing a FAR variant and by a corresponding host cell of the same type expressing the wild-type FAR from which the FAR variant may be derived are measured by culturing the cells under the same conditions, e.g, the same culture medium conditions (e.g., using LB or M9YE medium), the same temperature conditions (e.g., at 30° C. or at 37° C.), and for the same culture period conditions (e.g., culture for 24 hours).

In some embodiments, the engineered microorganisms of the present invention produce fatty alcohol compositions having an increased amount of C12:0 (1-dodecanol), an increased amount of C12:1 (cis Δ⁵-dodecenol), an increased amount of C14:0 (1-tetradecanol), an increased amount of C14:1 (cis Δ⁷-1-tetradecanol), an increased amount of C16:0 (1-hexadecanol), and/or an increased amount of C16:1 (cis Δ⁹-1-hexadecenol) relative to the wild-type FAR from which the FAR variant is derived. In some embodiments, the engineered microorganisms of the present invention produce fatty alcohol compositions having a decreased amount of C18:0 (1-octadecanol), and/or a decreased amount of C18:1 (cis Δ¹¹-1-octadecenol) relative to the wild-type FAR from which the FAR variant is derived.

In some embodiments, the fatty alcohol compositions that are produced comprise an increased amount of one or more of C12:0 (1-dodecanol), C12:1 (cis Δ⁵-dodecenol), C14:0 (1-tetradecanol), C14:1 (cis Δ⁷-1-tetradecanol), C16:0 (1-hexadecanol), and/or C16:1 (cis Δ⁹-1-hexadecenol) relative to the wild-type FAR from which the FAR variant is derived. For example, in some embodiments, the fatty alcohol compositions comprise an increased amount of C12:0 (1-dodecanol) relative to the wild-type FAR from which the FAR variant is derived (e.g., increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or more relative to the wild-type FAR enzyme from which the FAR variant is derived). In some embodiments, the fatty alcohol compositions comprise an increased amount of C12:1 (cis Δ⁵-dodecenol) relative to the wild-type FAR from which the FAR variant is derived (e.g., increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or more relative to the wild-type FAR enzyme from which the FAR variant is derived). In some embodiments, the fatty alcohol compositions comprise an increased amount of C14:0 (1-tetradecanol) relative to the wild-type FAR from which the FAR variant is derived (e.g., increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or more relative to the wild-type FAR enzyme from which the FAR variant is derived). In some embodiments, the fatty alcohol compositions comprise an increased amount of C14:1 (cis A⁷-1-tetradecanol) relative to the wild-type FAR from which the FAR variant is derived (e.g., increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or more relative to the wild-type FAR enzyme from which the FAR variant is derived). In some embodiments, the fatty alcohol compositions comprise an increased amount of C16:0 (1-hexadecanol) relative to the wild-type FAR from which the FAR variant is derived (e.g., increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or more relative to the wild-type FAR enzyme from which the FAR variant is derived). In some embodiments, the fatty alcohol compositions comprise an increased amount of C16:1 (cis Δ⁹-1-hexadecenol) relative to the wild-type FAR from which the FAR variant is derived (e.g., increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or more relative to the wild-type FAR enzyme from which the FAR variant is derived).

In some embodiments, the fatty alcohol compositions comprise a combination of two or more of: an increased amount of C12:0 (1-dodecanol) (e.g., increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or more relative to the wild-type FAR enzyme from which the FAR variant is derived); an increased amount of C12:1 (cis Δ⁵-dodecenol) (e.g., increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or more relative to the wild-type FAR enzyme from which the FAR variant is derived); an increased amount of C14:0 (1-tetradecanol) (e.g., increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or more relative to the wild-type FAR enzyme from which the FAR variant is derived); an increased amount of C14:1 (cis Δ⁷-1-tetradecanol) (e.g., increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or more relative to the wild-type FAR enzyme from which the FAR variant is derived); an increased amount of C16:0 (1-hexadecanol) (e.g., increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or more relative to the wild-type FAR enzyme from which the FAR variant is derived); an increased amount of C16:1 (cis Δ⁹-1-hexadecenol) (e.g., increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or more relative to the wild-type FAR enzyme from which the FAR variant is derived); a decreased amount of C18:0 (1-octadecanol) (e.g., decreased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or more relative to the wild-type FAR enzyme from which the FAR variant is derived); or a decreased amount of C18:1 (cis Δ¹¹-1-octadecenol) (e.g., decreased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or more relative to the wild-type FAR enzyme from which the FAR variant is derived).

In some embodiments, the fatty alcohol compositions produced by the engineered microorganisms and methods as described herein have a fatty alcohol profile comprising at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of C10 to C18 fatty alcohols.

In some embodiments, the fatty alcohol compositions produced by the engineered microorganisms and the methods described herein have a fatty alcohol profile comprising at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of C12 to C18 fatty alcohols.

In some embodiments, the fatty alcohol compositions produced by the engineered microorganisms and the methods described herein have a fatty alcohol profile comprising at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of C12 to C16 fatty alcohols (e.g., C12, C14, and C16 fatty alcohols).

In some embodiments, the fatty alcohol compositions produced by the engineered microorganisms and the methods described herein have a fatty alcohol profile comprising at least about 3%, at least about 4%, at least about 5%, at least about 8%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, or at least about 75% of C12 to C14 fatty alcohols (e.g., C12 and C14 fatty alcohols).

In some embodiments, the fatty alcohol compositions produced by the engineered microorganisms and the methods described herein have a fatty alcohol profile comprising at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of C14 to C16 (e.g., C14 and C16 fatty alcohols).

In some embodiments, the fatty alcohol compositions produced by the engineered microorganisms and the methods described herein have a fatty alcohol profile comprising at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, or at least about 75% of C12 fatty alcohols. In some embodiments, fatty alcohol compositions produced by the engineered microorganisms and the methods described herein have a fatty alcohol profile comprising from about 20% to about 60%, from about 20% to about 75%, from about 30% to about 60%, from about 30% to about 75%, from about 40% to about 60%, from about 40% to about 75% of C12 fatty alcohols.

In some embodiments, the fatty alcohol compositions produced by the engineered microorganisms and the methods described herein have a fatty alcohol profile comprising at least about 3%, at least about 4%, at least about 5%, at least about 8%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 45%, or at least about 50% of C14 fatty alcohols. In some embodiments, the fatty alcohol compositions produced by the engineered microorganisms and the methods described herein have a fatty alcohol profile comprising no more than about 40%, no more than about 35%, no more than about 30%, or no more than about 25% of C14 fatty alcohols. In some embodiments, the fatty alcohol compositions produced by the engineered microorganisms and the methods described herein have a fatty alcohol profile comprising from about 15% to about 40%, from about 20% to about 35%, or from about 20% to about 30% of C14 fatty alcohols.

In some embodiments, the fatty alcohol compositions produced by the engineered microorganisms and the methods described herein have a fatty alcohol profile comprising less than about 20%, less than about 15%, less than about 10%, or less than about 5% of C18 fatty alcohols.

In certain embodiments, the fatty alcohol compositions produced by the methods described herein have a fatty alcohol profile comprising a mixture of saturated and unsaturated C12 (e.g., C12:0 and C12:1), C14 (C14:0 and C14:1), C16 (C16:0 and C16:1), and C18 (C18:0 and C18-1) fatty alcohols. In some embodiments, the fatty alcohol profile comprises at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60% or more of C14:0 fatty alcohol. In some embodiments, the fatty alcohol profile further comprises at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, or more of C16:1 fatty alcohol. In some embodiments, the fatty alcohol profile further comprises at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% or more of C16:0 fatty alcohol. In some embodiments, the fatty alcohol profile further comprises up to about 1%, up to about 5%, up to about 10%, up to about 15%, up to about 20%, or up to about 25% of C18:1 fatty alcohol.

In some embodiments, the fatty alcohol profile comprises at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65% of C12 (e.g., C12:0 and/or C12:1) fatty alcohol. In some embodiments, the fatty alcohol profile further comprises at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, or more of C14 (e.g., C14:0 andor C14:1) fatty alcohol. In some embodiments, the fatty alcohol profile further comprises at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% or more of C16 (e.g., C16:0 and/or C16:1) fatty alcohol. In some embodiments, the fatty alcohol profile further comprises up to about 1%, up to about 5%, up to about 10%, up to about 15%, up to about 20%, or up to about 25% of C18 (e.g., C18:0 and/or C18:1) fatty alcohol.

Fatty Alcohol Measurements

FAR fatty alcohol production and fatty alcohol profiles (i.e., chain length distribution) can be determined by methods described in the Examples section and/or using any other method known in the art. Fatty alcohol production by an organism expressing a FAR variant can be described as an absolute quantity (e.g., moles/liter of culture) or as a fold-improvement over production by an organism or culture expressing a reference FAR sequence (e.g., a wild-type FAR or a different FAR variant).

Fatty alcohol production and/or fatty alcohol profiles by a microorganism expressing a FAR polypeptide can be measured, for example, using gas chromatography. In general, cells expressing a FAR variant are cultured, total or secreted fatty alcohols are isolated, and fatty alcohol amount and/or content is measured.

Any number of assays can be used to determine whether a host cell expressing a FAR variant as described herein produces an increased amount of fatty alcohols (e.g., at least 1.5 times more fatty alcohols) compared to a corresponding cell of the same type expressing a wild-type FAR, and/or whether a host cell expressing a FAR variant as described herein produces a different fatty alcohol profile compared to a corresponding cell of the same type expressing a wild-type FAR, including exemplary assays described herein. In one exemplary assay, fatty alcohols produced by productive E. coli strains are collected by extraction of 0.5 mL E. coli whole culture (culture medium plus cells) expressing a FAR variant using 1 mL of isopropanol:methyl t-butyl ether (MTBE) (4:6 ratio). The extraction mixture is allowed to shake for 2 hours at room temperature. The extraction mixture is then centrifuged, the upper organic phase transferred into a vial and analyzed by the gas chromatography (GC) equipped with flame ionization detector (FID) and DB-5MS column (length 30 m, I.D. 0.32 mm, film 0.25 um), starting at 150° C., and increasing the temperature at a rate of 25° C./min to 246° C., then holding for 1.81 min.

Fatty alcohol production by a host cell expressing a FAR variant can also be compared to a comparable cell (“control cell”) expressing a reference sequence, such as a wild-type FAR or a different FAR variant. Typically the FARs of the host and control cells are under control of the same promoter and the cells are maintained under the same conditions. For illustration, fatty alcohol production can be measured in E. coli (e.g., strain E. coli BW25113), using FARs under the control of the same promoter (e.g., the lac promoter), where the cells are cultured at 37° C. and fatty alcohol produced after 24 hours of culture are measured.

Fatty alcohol profiles (i.e., chain length distribution) can be determined, for example, using gas chromatography and/or mass spectroscopy. In an exemplary assay, fatty alcohols are produced as described above and the identification of individual fatty alcohols is performed by comparison to commercial standards (Sigma Chemical Company, 6050 Spruce St. Louis, Mo. 63103). The identity of the peaks can also be confirmed by running the samples through a gas chromatography (GC) equipped with mass spectrometer (MS) as needed.

IV. Far Variants

In one aspect, the sequences of the improved FAR polypeptides described herein comprise at least about 70% sequence identity with a wild-type FAR polypeptide and comprise one or more mutations (e.g. amino acid substitutions) as compared to the wild-type FAR from which the FAR variant is derived, such that when expressed in a recombinant host cell, the cell produces an increased amount of fatty alcohols or produces a fatty alcohol composition comprising changes in a fatty alcohol profile as compared to a control cell expressing a wild-type FAR. Substitutions that yield increased fatty alcohol production under these conditions are described herein. These substitutions can be used singly or in any combinations. In some embodiments, a FAR variant comprises a single substitution set, e.g., a substitution set of any of Tables 1, 2, 4, 7, 8, 9, 10, or 11. In some embodiments, a FAR variant comprises two or more (e.g., two, three, four, five, or more) substitution sets. In some embodiments, combinations of substitutions can be selected so as to provide fatty alcohol production under the conditions specified that is at least about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold greater than that of the wild-type FAR (e.g., SEQ ID NO:2 or SEQ ID NO:4). In some embodiments, combinations of substitutions can be selected so as to provide fatty alcohol compositions having increased amounts of C12 fatty alcohols, C14 fatty alcohols, and/or C16 fatty alcohols under the conditions specified as compared to the wild-type FAR (e.g., SEQ ID NO:2 or SEQ ID NO:4). In some embodiments, combinations of substitutions can be selected so as to provide fatty alcohol compositions wherein an increased proportion of the total composition comprises C12 fatty alcohols, C14 fatty alcohols, and/or C16 fatty alcohols under the conditions specified as compared to the wild-type FAR (e.g., SEQ ID NO:2 or SEQ ID NO:4).

It has been discovered that certain substitutions, both singly and in various combinations, yield an increase in the shorter chain (e.g., C12 and C14) fatty alcohols produced by an engineered microorganism (e.g., an increased total amount of C12 and C14 fatty alcohols and/or an increased proportion of C12 and C14 fatty alcohols in a fatty alcohol composition), relative to a wild-type FAR or a reference FAR variant. These substitutions may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more than 30 substitutions. In some embodiments, the FAR variants of the invention differ from wild-type FAR (e.g., SEQ ID NO:2 or SEQ ID NO:4) or a reference FAR variant (e.g., SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:14) by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more amino acid residues. In some embodiments, the FAR variant polypeptides of the invention differ from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:14 in up to 20 residues. In some embodiments, the improved FAR polypeptides differ from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:14 in up to 15 residues, sometimes in up to 12 residues, or sometimes in up to 10 residues.

As shown herein, for example in Tables 1, 2, 4, 7, 8, 9, 10, and 11, it can be seen that substitutions introduced at any of a number of different amino acid positions in the wild-type FAR of SEQ ID NO:2 or the wild-type FAR of SEQ ID NO:4 have yielded FAR variants capable of producing fatty alcohol compositions with increased levels of shorter chain fatty alcohols (e.g., C12 and C14) when introduced into a host cell or microorganism (e.g., a bacteria, yeast, filamentous fungi, or algae), as compared to the fatty alcohol chain length composition produced by the wild-type FAR when introduced into corresponding host cells or microorganisms of the same type. In addition, substitutions (e.g., substitutions listed in any of Tables 1, 2, 4, 7, 8, 9, 10, or 11) that are introduced into a FAR variant reference sequence, such as SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:14, have yielded FAR variants capable of producing fatty alcohol compositions with increased levels of shorter chain fatty alcohols (e.g., C12 and C14) when introduced into a host cell or microorganism, as compared to the fatty alcohol chain length composition produced by the FAR variant reference sequence when introduced into corresponding host cells or microorganisms of the same type. These substitutions may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more than 30 substitutions.

In some embodiments, a FAR variant polypeptide comprises at least about 75% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:2 (wild-type M. algicola FAR) and also comprises one or more amino acid substitutions as described herein (e.g., one or more amino acid substitutions or amino acid substitution sets listed in Table 1, Table 2, Table 4, Table 7, Table 8, Table 9, or Table 10).

In some embodiments, a FAR variant polypeptide comprises at least about 75% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:4 (wild-type M. aquaeolei FAR) and also comprises one or more amino acid substitutions as described herein (e.g., one or more amino acid substitutions or amino acid substitution sets listed in Table 11).

In some embodiments, a FAR variant polypeptide comprises at least about 75% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to a reference FAR polypeptide (e.g., a FAR variant of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:14) and also comprises one or more amino acid substitutions as described herein. In some embodiments, said FAR variant polypeptide comprises an amino acid substitution set listed in Table 1, Table 2, Table 4, Table 7, Table 8, Table 9, Table 10, or Table 11. In another aspect, the invention relates to FAR variants that comprise an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions over substantially the entire length of a nucleic acid corresponding to SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9. SEQ ID NO:11, or SEQ ID NO:13, wherein the FAR variant, when expressed in a recombinant host cell, produces an increased amount of fatty alcohols or produces a fatty alcohol composition comprising changes in a fatty alcohol profile as compared to a control cell expressing the reference FAR polypeptide. Exemplary reference FAR polypeptides include, for illustration and not limitation, SEQ ID NOs:6, 8, 10, 12, and 14.

In certain embodiments the FAR variant comprises an amino acid sequence having at least 80% (alternatively, at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:2 and comprises a substitution at one or more positions selected from positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 23, 40, 43, 44, 45, 47, 49, 50, 52, 61, 62, 63, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 83, 84, 85, 86, 87, 88, 89, 90, 91, 93, 97, 98, 100, 101, 103, 104, 106, 107, 108. 111, 112, 113, 115, 116, 118, 120, 121, 122, 123, 126, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 140, 144, 145, 148, 150, 151, 153, 154, 155, 156, 157, 158, 160, 161, 162, 163, 164, 166, 167, 174, 176, 177, 178, 179, 180, 181, 182, 185, 186, 187, 188, 189, 190, 191, 192, 193, 195, 196, 197, 198, 200, 205, 206, 207, 208, 209, 211, 212, 215, 216, 218, 220, 221, 224, 225, 226, 227, 228, 231, 235, 236, 238, 239, 240, 241, 242, 244, 245, 246, 247, 253, 258, 263, 264, 266, 267, 268, 270, 273, 275, 277, 278, 280, 281, 283, 284, 285, 286, 288, 303, 306, 308, 310, 313, 316, 318, 331, 337, 338, 339, 341, 351, 352, 355, 359, 361, 362, 363, 365, 368, 370, 373, 374, 376, 377, 380, 382, 384, 387, 388, 389, 393, 396, 397, 398, 399, 400, 401, 402, 404, 405, 406, 308, 409, 410, 411, 412, 413, 414, 416, 417, 418, 419, 421, 424, 426, 429, 430, 432, 433, 436, 442, 443, 446, 452, 458, 463, 465, 466, 474, 478, 482, 487, 489, 490, 491, 494, 495, 496, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, or 512, wherein the position is numbered with reference to SEQ ID NO:2. In some embodiments, the FAR variant comprises substitutions at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more positions. In certain embodiments, the FAR variant comprises an amino acid sequence having at least 80% (alternatively, at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:2 and comprises one or more substitutions selected from M1E/G/L/RN/W, A2D/F/G/H/P/Q/R/S/T/W/Y, T3/I/L, Q4/N/R/S/W/Y, Q5M/N, Q6C/H/K/P/R/SN/Y, Q7H/N, N8A/E/HN, G9C/FN, A10T, S11D/G, A12D/R/S/T, S13G/L/V, G14K/L/M/RN, V151, L16G/I/S, E17C/G/H/R, Q181/L, R20K, H23R, K40R, R43H, T44A, V45A/S, D47E/N, G49E, G50A, H52Y, H61R, P62Q, A63D, R65G, E66D/F/S/Y, F68AN, L69E/I/M/Q, N70D/E/L/M/R/T, E71C/M/Q/S, 172L, A73G/H/K/L/M, S74K/L/P/T/W, S75C/E/H/N, S76E/F/I/L/R, V77I/P/T, F78M, E79D/I/L/Q/V, R801/L, L81F/T, H83E, D84A, D85E, N86S, E87V, A88G/V, F89D/N/P/R, E90D/Q, T91A/R, L93D, V97I, H98P, 1100V, T101A, E103C/S/V, V104I/M, E106A/H, S107A, R108E/G/H/Q, L111I, T112G, P113I/Q, R115D, F116Y, A118K, A120C, G121T, Q122E/H/T, V123L, F126V, N128C/H/L, S129D, A130C/S, A131P/S, S132H, V133A/G, N134D/K/R/S/V, F135E, R136L, E137L, E138Q/R, D140Y, K144A/E/R, I145E/H, L148E/K/T, L150P, E151G/RN, V153F/I, A154G/R, A155G/M/R/T/W, L156M, A157Q/V, E158D/N, N160T, S161P/Y, A162K, M163L, A164V, 1166L/M, Q167H, N174A, K176G/I/M, N177D/E/L/R/T, S178F/L, G179D/S/W, Q180C/R, 1181D/E/L/V, T182G/I/K/R, V185G/1/P, I186H, K187P, P188D/E/I/R/S/W, A189L/N, G190I/K/L, E191V/W, S192A, I193C/L/V, R195F/H/I/N/W, S196D, T197F/P, D198S, Y200F, E205K, L206C, V207L/M, H208R, L209N/T/Y, Q211H/L/N/R, D212F, S215EN, D216G/Q, K218P/Q/R, R220A/H, Y221D/K, K224R, V225C/M, L226M, E227G, K228H, V231A, 1235E, R236I, A238G, N239C, N240Q/R/T, Y241F, G242E, S244A/P/R, D245H, T246A/P/V, Y247N, L253P/V, L258P, S263N, G264R, S266A/T, L267H, T268N, V270L, S273F, 1275V, S277A, A278C, E2801, E281S/Y, S283A/E/F/M/T, P284C/L/Q, G285D, W286Y, E288D/H/Q, E303G, S306T/W, F3081, G310L/V, S313Q, I316L, V318F/L/M, S331V, S337G, G338E, S339P, Q341R, G351C, S352G, I355F/L/S/W, K359E, I361C/F/L, D362L, Y363H, M365N, A368S, T370A/I, A373W, A374Y, D376K/P/R, Q377H/K, Y380H/R, R382H/Q, T384S, F3871/L, V388L, A3891/M/L/V, K393A, D396G, V397L, V398Y, V399I, G400S, G401A/C/L/S/TN, M402V, V404I/L, P405A/C/F/G/L/V, L406Y, I408L, A409T/V/W/Y, G410D/H/R, K411R, A412V, M413L/R, R414K, A416L/V, G417V, Q418I/N/RN, N419S, E421D/G/I/L/P/R/S/V, V424M, K426R/T, D429E/K/R, T430A/H/I, R432C/Q, S433H/K/N/R/Y, T436A, T442I, A443T, Y446F, S452E/G/N, S458E/N/Q, L463V, R465K, V466G/Q, Q474L/R, Q478E, C482R, G487R/Y, L489F, N490C/S, R491M, L494Y, K495C/S, E496D/G, K498G/N, L499R/S/V, Y500H/N, S501C/F/W, L502A/Q/R/S/W, R503c/K, A504D/E/S/T, A505E/G/K, D506G/L/M/R/W, T507H/Q, R508D/G/H/L/M, K509D/G/H/P/Q/R, K510D/E/L/M/N/R/S, A511D/G/I/K/P/R/S/T, and/or A512M/R. In some embodiments, the FAR variant comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more substitions.

In some embodiments, the FAR variant comprises an amino acid sequence having at least 80% (alternatively, at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:4 and comprises a substitution at one or more positions selected from positions 8, 74, 116, 135, 228, 411, 430, 434, 438, 503, 512, and 513, wherein the position is numbered with reference to SEQ ID NO:4. In some embodiments, the FAR variant comprises substitutions at 2, 3, 4, 5 or more positions. In certain embodiments, the FAR variant comprises an amino acid sequence having at least 80% (alternatively, at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:4 and comprises one or more substitutions selected from H8K, A74K/L, D116A/E, N135K, E228G, D411R, D430K, S434K/F/W, I438V, L503R/S, A512G/Q/P, and A513G/K/R/S/T/P. In some embodiments, the FAR variant comprises 2, 3, 4, 5 or more substitutions.

In certain embodiments, the FAR variant comprises an amino acid sequence having at least 90% (alternatively, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:6 and comprises a substitution at one or more positions selected from positions 14, 18, 65, 69, 71, 74, 77, 87, 91, 98, 104, 128, 134, 137, 138, 148, 153, 161, 180, 185, 188, 207, 209, 224, 227, 244, 246, 266, 283, 288, 303, 306, 331, 351, 365, 370, 374, 376, 377, 380, 382, 389, 398, 401, 404, 405, 406, 409, 410, 412, 413, 416, 418, 421, 429, 430, 432, 433, 443, 446, 452, 458, 466, 474, 487, 499, 500, 502, 505, 508, 509, 510, and 511, wherein the position is numbered with reference to SEQ ID NO:6. In certain embodiments, the FAR variant comprises an amino acid sequence having at least 80% (alternatively, at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:6 and comprises a substitution at one or more positions selected from 14R/V, 18Q, 61R, 65G, 69E/Q, 71Q, 74K/P, 77I, 87V, 91R, 98P, 104I/M, 128H, 134R/K/S, 137L, 138Q, 148E, 153I, 161P, 180R, 185I, 188I, 207L, 209N/T, 224R, 227G, 244A/P, 246A, 266A, 283M/F/E/T, 288Q, 303G, 306W, 331V, 351C, 365N, 370I, 374Y, 376P, 377K, 380R, 382H, 389I/M/L/V, 398Y, 401L/V/S/A/C, 405L/C/V/A/F/G, 404I, 406Y, 409V/W/Y, 410H/R, 412V, 413R, 416L/V, 418R/V/I, 421R/I/S/L/N/V/P, 429K/R/E, 430I/H, 432C/Q, 433H/N/K/Y/R, 443T, 446F, 452N/G, 458Q, 466Q, 474R, 487R/Y, 499S, 500N, 502R/S/A/Q/, 505K, 508G/H/D, 509H/D, 510D, and/or 511S/K/T/R/G. In some embodiments, the FAR variant comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more substitutions.

In certain embodiments, the FAR variant comprises an amino acid sequence having at least 90% (alternatively, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:8 and comprises a substitution at one or more positions selected from positions 18, 61, 65, 74, 77, 104, 134, 137, 161, 246, 266, 283, 306, 370, 374, 380, 382, 389, 398, 401, 405, 410, 412, 421, 429, 446, 487, 499, 502, 505, 508, and 511, wherein the position is numbered with reference to SEQ ID NO:8. In certain embodiments, the FAR variant comprises an amino acid sequence having at least 90% (alternatively, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:8 and comprises one or more substitutions selected from 18Q, 61R, 65R, 74P, 771/Q, 1041, 134R/K, 137L, 161P, 246A, 266A, 283F, 306W, 370I, 374Y, 380R, 382H, 389M, 398Y, 401V, 405C/L/A, 410R, 412V, 421G/S/R, 429K, 446F, 487Y/G, 499R/S, 502W, 505K, 508G, and 511K. In some embodiments, the FAR variant comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more substitutions.

In certain embodiments, the FAR variant comprises an amino acid sequence having at least 95% (alternatively, at least 96%, at least 97%, at least 98%, at least 99% and even 100%) sequence identity to SEQ ID NO:10 and comprises a substitution at one or more positions selected from positions 18, 61, 65, 74, 77, 104, 134, 137, 161, 246, 266, 283, 306, 370, 374, 380, 382, 389, 398, 401, 405, 410, 412, 421, 429, 446, 487, 499, 502, 505, 508, and 511, wherein the position is numbered with reference to SEQ ID NO:10. In some embodiments, the FAR variant comprises an amino acid sequence having at least 95% (alternatively, at least 96%, at least 97%, at least 98%, at least 99% and even 100%) sequence identity to SEQ ID NO:10 and comprises one or more substitutions selected from 18Q, 61R, 65R, 74P, 771/Q, 104I, 134R/K, 137L, 161P, 246A, 266A, 283F, 306W, 370I, 374Y, 380R, 382H, 389M, 398Y, 401V, 405C/L/A, 410R, 412V, 421G/S/R, 429K, 446F, 487Y/G, 499R/S, 502W, 505K, 508G, and 511K. In some embodiments, the variant FAR comprises an amino acid sequence having at least 95% (alternatively, at least 96%, at least 97%, at least 98%, at least 99% and even 100%) sequence identity to SEQ ID NO:10 and comprises a substitution at at least two, three, four, or more positions, wherein at least two of the positions are selected from 18, 61, 65, 74, 77, 104, 134, 137, 161, 246, 266, 283, 306, 370, 374, 380, 382, 389, 398, 401, 405, 410, 412, 421, 429, 446, 487, 499, 502, 505, 508, and 511. In some embodiments, the variant FAR comprises an amino acid sequence having at least 95% (alternatively, at least 96%, at least 97%, at least 98%, at least 99% and even 100%) sequence identity to SEQ ID NO:10 and comprises two, three, four, or more substitutions, wherein at least two of the positions are selected from positions 18Q, 61R, 65R, 74P, 77I/Q, 104I, 134R/K, 137L, 161P, 246A, 266A, 283F, 306W, 370I, 374Y, 380R, 382H, 389M, 398Y, 401V, 405C/L/A, 410R, 412V, 421G/S/R, 429K, 446F, 487Y/G, 499R/S, 502W, 505K, 508G, and 511K. In some embodiments, the FAR variant comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more substitutions.

In some embodiments, a FAR variant comprises a substitution at one or more positions selected from positions 134, 138, 188, 405, 418, 458, 502, 508, 509, and 511, wherein the position is numbered with reference to SEQ ID NO:2. In some embodiments, a FAR variant comprises one or more substitutions selected from N134S, E138Q, P188S, P405V, Q418V, S458Q, L502S, R508D, K509D, and A511T. In some embodiments, a FAR variant comprises the substitution set N134S, E138Q, P1885, P405V, Q418V, S458Q, L502S, R508D, K509D, and A511T. In some embodiments, a FAR variant has the amino acid sequence of SEQ ID NO:8.

In some embodiments, a FAR variant comprises a substitution at one or more positions selected from positions 18, 65, 128, 134, 138, 177, 188, 224, 226, 405, 418, 433, 458, 487, 502, 508, 509, and 511, wherein the position is numbered with reference to SEQ ID NO:2. In some embodiments, a FAR variant comprises one or more substitutions selected from Q181, R65G, N128H, N134S, E138Q, N177T, P188S, K224R, L226M, P405V, Q418V, S433K, S458Q, G487R, L502S, R508D, K509D, and A511T. In some embodiments, a FAR variant comprises the substitution set Q181, R65G, N128H, N134S, E138Q, N177T, P188S, K224R, L226M, P405V, Q418V, S433K, S458Q, G487R, L502S, R508D, K509D, and A511T. In some embodiments, a FAR variant has the amino acid sequence of SEQ ID NO:10.

In some embodiments, a FAR variant comprises a substitution at one or more positions selected from positions 7, 18, 65, 128, 138, 177, 188, 224, 226, 227, 365, 401, 405, 418, 433, 458, 487, 502, 508, 509, and 511, wherein the position is numbered with reference to SEQ ID NO:2. In some embodiments, a FAR variant comprises one or more substitutions selected from Q7N, Q181, R65G, N128H, E138Q, N177T, P188S, K224R, L226M, E227G, M365N, G401V, P405V, Q418V, S433K, S458Q, G487R, L5025, R508D, K509D, and A511T. In some embodiments, a FAR variant comprises the substitution set Q7N, Q181, R65G, N128H, E138Q, N177T, P188S, K224R, L226M, E227G, M365N, G401V, P405V, Q418V, S433K, S458Q, G487R, L502S, R508D, K509D, and A511T. In some embodiments, a FAR variant has the amino acid sequence of SEQ ID NO:16.

In some embodiments, a FAR variant comprises a substitution at one or more positions selected from positions 7, 18, 65, 104, 128, 138, 177, 188, 224, 226, 227, 365, 401, 405, 410, 418, 433, 458, 487, 502, 508, 509, and 511, wherein the position is numbered with reference to SEQ ID NO:2. In some embodiments, a FAR variant comprises one or more substitutions selected from Q7N, Q181, R65G, V104I, N128H, E138Q, N177T, P188S, K224R, L226M, E227G, M365N, G401V, P405V, G410R, Q418V, S433K, S458Q, G487R, L502S, R508D, K509D, and A511T. In some embodiments, a FAR variant comprises the substitution set Q7N, Q181, R65G, V104I, N128H, E138Q, N177T, P188S, K224R, L226M, E227G, M365N, G401V, P405V, G410R, Q418V, S433K, S458Q, G487R, L502S, R508D, K509D, and A511T. In some embodiments, a FAR variant has the amino acid sequence of SEQ ID NO:18.

In some embodiments, a FAR variant comprises a substitution at one or more positions selected from positions 7, 18, 65, 104, 128, 138, 177, 188, 224, 226, 227, 318, 365, 401, 405, 410, 418, 433, 458, 487, 502, 508, 509, and 511, wherein the position is numbered with reference to SEQ ID NO:2. In some embodiments, a FAR variant comprises one or more substitutions selected from Q7N, Q181, R65G, V104I, N128H, E138Q, N177T, P188S, K224R, L226M, E227G, V318F, M365N, G401V, P405V, G410R, Q418V, S433K, S458Q, G487R, L502S, R508D, K509D, and A511T. In some embodiments, a FAR variant comprises the substitution set Q7N, Q181, R65G, V104I, N128H, E138Q, N177T, P188S, K224R, L226M, E227G, V318F, M365N, G401V, P405V, G410R, Q418V, S433K, S458Q, G487R, L502S, R508D, K509D, and A511T. In some embodiments, a FAR variant has the amino acid sequence of SEQ ID NO:20.

In some embodiments, a FAR variant comprises a substitution at one or more positions selected from positions 7, 18, 104, 128, 138, 177, 188, 224, 226, 227, 318, 361, 365, 401, 405, 410, 418, 433, 458, 487, 496, 502, 508, 509, 510, and 511, wherein the position is numbered with reference to SEQ ID NO:2. In some embodiments, a FAR variant comprises one or more substitutions selected from Q7N, Q181, V104I, N128H, E138Q, N177T, P188S, K224R, L226M, E227G, V318F, I361F, M365N, G401V, P405V, G410R, Q418V, S433K, S458Q, G487R, E496G, L502S, R508D, K509D, K510E, and A511T. In some embodiments, a FAR variant comprises the substitution set Q7N, Q181, V104I, N128H, E138Q, N177T, P188S, K224R, L226M, E227G, V318F, I361F, M365N, G401V, P405V, G410R, Q418V, S433K, S458Q, G487R, E496G, L502S, R508D, K509D, K510E, and A511T. In some embodiments, a FAR variant has the amino acid sequence of SEQ ID NO:22.

In some embodiments, a FAR variant comprises a substitution at one or more positions selected from positions 7, 14, 18, 104, 128, 138, 177, 188, 189, 220, 224, 226, 227, 316, 318, 355, 361, 365, 401, 405, 410, 418, 433, 458, 487, 496, 502, 508, 509, 510, and 511, wherein the position is numbered with reference to SEQ ID NO:2. In some embodiments, a FAR variant comprises one or more substitutions selected from Q7N, G14L, Q181, V104I, N128H, E138Q, N177T, P188S, A189N, R220H, K224R, L226M, E227G, I316L, V318F, I355F, I361F, M365N, G401V, P405V, G410H, Q418V, S433K, S458Q, G487R, E496G, L502A, R508D, K509D, K510E, and A511T. In some embodiments, a FAR variant comprises the substitution set Q7N, G14L, Q181, V104I, N128H, E138Q, N177T, P188S, A189N, R220H, K224R, L226M, E227G, I316L, V318F, I355F, I361F, M365N, G401V, P405V, G410H, Q418V, S433K, S458Q, G487R, E496G, L502A, R508D, K509D, K510E, and A511T. In some embodiments, a FAR variant has the amino acid sequence of SEQ ID NO:24.

In some embodiments, a FAR variant comprises a substitution at one or more positions selected from positions 6, 7, 18, 104, 108, 128, 138, 177, 188, 224, 226, 227, 318, 355, 361, 365, 401, 405, 410, 418, 433, 458, 487, 496, 502, 508, 509, 510, and 511, wherein the position is numbered with reference to SEQ ID NO:2. In some embodiments, a FAR variant comprises one or more substitutions selected from Q6R, Q7N, Q181, V104I, R108H, N128H, E138Q, N177T, P188S, K224R, L226M, E227G, V318F, I355L, I361F, M365N, G401V, P405V, G410R, Q418V, S433K, S458Q, G487R, E496G, L502S, R508D, K509D, K510E, and A511T. In some embodiments, a FAR variant comprises the substitution set Q6R, Q7N, Q181, V104I, R108H, N128H, E138Q, N177T, P188S, K224R, L226M, E227G, V318F, I355L, I361F, M365N, G401V, P405V, G410R, Q418V, S433K, S458Q, G487R, E496G, L502S, R508D, K509D, K510E, and A511T. In some embodiments, a FAR variant has the amino acid sequence of SEQ ID NO:26.

In some embodiments, a FAR variant comprises a substitution at one or more positions selected from positions 4, 6, 7, 18, 62, 104, 108, 128, 138, 177, 188, 224, 226, 227, 316, 318, 355, 361, 365, 401, 405, 410, 418, 433, 458, 487, 496, 502, 507, 508, 509, 510, and 511, wherein the position is numbered with reference to SEQ ID NO:2. In some embodiments, a FAR variant comprises one or more substitutions selected from Q4Y, Q6R, Q7N, Q181, P62Q, V104I, R108H, N128H, E138Q, N177T, P188S, K224R, L226M, E227G, I316L, V318F, I355L, I361F, M365N, G401V, P405V, G410H, Q418V, S433K, S458Q, G487R, E496G, L502S, T507H, R508D, K509D, K510E, and A511I. In some embodiments, a FAR variant comprises the substitution set Q4Y, Q6R, Q7N, Q181, P62Q, V104I, R108H, N128H, E138Q, N177T, P188S, K224R, L226M, E227G, I316L, V318F, I355L, I361F, M365N, G401V, P405V, G410H, Q418V, S433K, S458Q, G487R, E496G, L502S, T507H, R508D, K509D, K510E, and A511I. In some embodiments, a FAR variant has the amino acid sequence of SEQ ID NO:28.

In certain embodiments, the FAR variant comprises an amino acid sequence having at least 80% (alternatively, at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:2 and comprises one or more substitution sets selected from the substitutions sets listed in any of Table 1, Table 2, Table 4, Table 7, Table 8, Table 9, Table 10, or Table 11. In some embodiments, the FAR variant comprises at least 80% sequence identity to SEQ ID NO:2 and comprises one or more substitution sets selected from the substitutions sets listed in any of Table 1, Table 2, Table 4, Table 7, Table 8, Table 9, Table 10, or Table 11, and a host cell or microorganism expressing the FAR variant produces an increased amount of shorter chain fatty alcohols (e.g., C12 and C14) as compared to a host cell or microorganism expressing a wild-type FAR of SEQ ID NO:2. In some embodiments, a host cell or microorganism expressing the FAR variant produces at least 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold or more of C12 and C14 fatty alcohols as compared to a host cell or microorganism expressing a wild-type FAR of SEQ ID NO:2. In some embodiments, the fatty alcohol composition comprising C12 and C14 fatty alcohols produced from a host cell or microorganism comprising the FAR variant will comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% C12 and C14 fatty alcohols.

In certain embodiments, the FAR variant comprises an amino acid sequence having at least 80% (alternatively, at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:4 and comprises one or more substitution sets selected from the substitutions sets listed in any of Table 1, Table 2, Table 4, Table 7, Table 8, Table 9, Table 10, or Table 11. In some embodiments, the FAR variant comprises one or more substitution sets listed in Table 11. In some embodiments, the FAR variant comprises at least 80% sequence identity to SEQ ID NO:4 and comprises one or more substitution sets selected from the substitutions sets listed in any of Table 1, Table 2, Table 4, Table 7, Table 8, Table 9, Table 10, or Table 11, and a host cell or microorganism expressing the FAR variant produces an increased amount of shorter chain fatty alcohols (e.g., C12 and C14) as compared to a host cell or microorganism expressing a wild-type FAR of SEQ ID NO:4. In some embodiments, a host cell or microorganism expressing the FAR variant produces at least 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold or more of C12 and C14 fatty alcohols as compared to a host cell or microorganism expressing a wild-type FAR of SEQ ID NO:4. In some embodiments, the fatty alcohol composition comprising C12 to C14 fatty alcohols produced from a host cell or microorganism comprising the FAR variant will comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% C12 and C14 fatty alcohols.

Improved or increased fatty alcohol production of a FAR variant relative to a reference polypeptide can be detected by comparing fatty alcohol production by host cells expressing the FAR variant to fatty alcohol production by host cells (of the same type) expressing the reference protein (which may be a wild-type or variant FAR) or a host cell not expressing exogenous FAR. It will be understood by those of skill that it is desirable that the only paramater varied between the cells is the FAR being expressed (e.g., a wild-type FAR or a FAR variant). Thus, for example, the FAR variant and reference polypeptide will be encoded by polynucleotides with the same sequence except at codons corresponding to substitutions (typically a sequence that is codon optimized for the cell type) and will be controlled by the same promoter, and cells expressing the polypeptides will be cultured under the same conditions. Improved or increased fatty alcohol production of a cell expressing a FAR variant relative to a cell not expressing an exogenous FAR may also be measured.

For bacterial host cells, such as E. coli, exemplary assay conditions are described in Examples 2 and 4. In one approach, E. coli are transformed with an expression cassette comprising a sequence encoding the FAR variant or the reference protein (e.g., wild-type FAR, such as FAR Maa or FAR Maq) and an operably linked promoter. The cells may be stably transformed. The promoter may be constitutive or inducible. The lac promoter may be used. The cells are grown in medium (e.g., M9YE medium containing 0.5-5% glucose; Dunny, G. M., and Clewell, D. B., 1975. J. Bacteriol. 124:784-790) and any appropriate selection agents (see Examples 2 and 4). In one approach the cells are cultured for a period of time (e.g., 18 hours or 24 hours) and fatty alcohol production is assayed. Typically, total fatty alcohol is assayed, but the amount of fatty alcohol secreted into the medium may be assayed if desired. In some embodiments, the promoter is inducible. For example, cells may be grown (e.g., to an OD₆₀₀ of 0.6-0.8), at which point expression of the heterologous FAR gene is induced (e.g., by addition of IPTG to a 1 mM final concentration when the lac promoter is used). Incubation is continued for 24 hours at 30° C. (alternatively 37° C. or 40° C.) and fatty alcohol production is assayed.

Other exemplary assays for assessing FAR activity are known in the art. See, for example, U.S. Pat. No. 5,370,996; US 2010/0203614; and Wahlen et al., Appl. Environ. Microbiol. 75(9):2758 (2009).

It will be apparent that the same assays may be used to asssess FAR activity of functionally active fragments of FAR variants.

Functional Fragments of FAR Variants

Skilled artisans will appreciate that oftentimes, the full length sequence of an enzyme is not required for enzymatic activity. Therefore, “functional fragments” of the various FAR variants described herein are also contemplated and included in the disclosure. In some embodiments the term “FAR variants” encompasses functional fragments. Sometimes both FAR variants and functional fragments thereof are referred to herein as “improved FAR variant polypeptides.” The enzymatic activity of functional fragments may be measured as described in the hereinbelow.

In some embodiments the functional fragments comprise at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98% and at least 99% of the corresponding full-length FAR variant enzyme (e.g., a FAR variant of any of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, or SEQ ID NO:28). In many instances, functional fragments, like the full-length FAR variant enzyme from which they are derived, will produce 1.5-fold or higher total fatty alcohol than the wild-type FAR of SEQ ID NO:2, when assayed under the same conditions. In some embodiments, the functional fragments of the FAR variants of the invention are from about 350 to about 550 amino acids in length, e.g., from about 350 to about 500 amino acids in length, from about 350 to about 475 amino acids in length, from about 350 to about 450 amino acids in length and also from about 350 to about 400 amino acids in length. In some embodiments, the functional fragments of FAR variants of the invention are from 400 to 550 amino acids in length, such as from about 400 to 500, from about 450 to 500, from about 475 to 500, from about 500 to 525, or from 505 to 515 amino acids in length. In some embodiments, the functional fragments of the FAR variants of the invention are from about 350 to about 550 amino acids in length, e.g., from about 350 to about 500 amino acids in length, from about 350 to about 475 amino acids in length, from about 350 to about 450 amino acids in length and also from about 350 to about 400 amino acids in length. In some embodiments, the functional fragments of FAR variants of the invention are from 400 to 550 amino acids in length, such as from about 400 to 500, from about 450 to 500, from about 475 to 500, from about 500 to 525, or from 505 to 515 amino acids in length as compared to a full-length FAR variant (e.g., a FAR variant of any of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, or SEQ ID NO:28).

In some embodiments, regions of one or both termini, such as, for example, from about 1 to about 10; 1 to about 15; or 1 to about 20 residues at one or both termini, may be removed without significantly deleteriously affecting the activity of the enzyme. Such deletions can often times be made internally without detrimental effect.

Exemplary Substitutions in Marinobacter algicola FAR Homologs

In another aspect, the present invention provides FAR variants of naturally occurring FAR enzymes of marine proteobacteria species other than Marinobacter algicola (strain DG893) which comprise a substitution or modification at least one position corresponding to a substitution of a M. algicola FAR variant described herein, and which have improved properties relative to the naturally occurring FAR enzyme.

In particular, analogous substitutions may be made in marine gammaproteobacteria with significant sequence similarity to Marinobacter algicola (strain DG893). For example, analogous substitutions may be made in other species of Marinobacter including but not limited to M. algicola, M. aquaeolei, M. arcticus, M. actinobacterium, and M. lipolyticus; species of Oceanobacter including but not limited to Oceanobacter sp. Red65 (renamed Bermanella marisrubi), Oceanobacter strain WH099, and O. kriegii; and species of Hahella including but not limited to H. chejuensis and equivalent species thereof.

It is within the ability of one of ordinary skill in the art to identify other examples of structurally homologous proteins. The present invention provides variants of these and other FAR proteins in which substitutions are made at residues corresponding to those identified herein in the M. algicola FAR protein.

To produce FAR homologs with improved properties, the sequences of the wild-type M. algicola FAR and the FAR homolog (e.g., a M. aquaeolei FAR protein) can be aligned in a pairwise manner as described supra. Based on the alignment, a residue in a position in the homolog that corresponds, based on the alignment, with a specified position in M. algicola FAR is identified. For example, analogous substitutions in wild-type M. aquaeolei FAR to those substitutions described in Table 1, Table 2, Table 4, Table 7, Table 8, Table 9, or Table 10 can be made by aligning the wild-type M. algicola FAR protein (SEQ ID NO:2) with the wild-type M. aquaeolei FAR protein (SEQ ID NO:4). Analogous substitutions in M. aquaeolei are described herein in Example 10.

Thus, in some embodiments, the present invention provides a recombinant FAR variant comprising at least about 70% (or at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%) sequence identity to SEQ ID NO:2 and comprising one or more amino acid substitutions selected from X1E/G/URN/W, X2D/F/G/H/P/Q/R/S/T/W/Y, X3/I/L, X4/N/R/S/W/Y, X5M/N, X6C/H/K/P/R/S/V/Y, X7H/N, X8A/E/H/V, X9C/FN, X10T, X11D/G, X12D/R/S/T, X13G/L/V, X14K/L/M/R/V, X15I, X16G/I/S, X17C/G/H/R, X18I/L, X20K, X23R, X40R, X43H, X44A, X45A/S, X47E/N, X49E, X50A, X52Y, X61R, X62Q, X63D, X65G, X66D/F/S/Y, X68A/V, X69E/I/M/Q, X70D/E/L/M/R/T, X71C/M/Q/S, X72L, X73G/H/K/L/M, X74K/L/P/T/W, X75C/E/H/N, X76E/F/I/L/R, X77I/P/T, X78M, X79D/I/L/Q/V, X80I/L, X81F/T, X83E, X84A, X85E, X86S, X87V, X88G/V, X89D/N/P/R, X90D/Q, X91A/R, X93D, X97I, X98P, X100V, X101A, X103C/S/V, X104I/M, X106A/H, X107A, X108E/G/H/Q, X1111, X112G, X113I/Q, X115D, X116Y, X118K, X120C, X121T, X122E/H/T, X123L, X126V, X128C/H/L, X129D, X130C/S, X131P/S, X132H, X133A/G, X134D/K/R/S/Y, X135E, X136L, X137L, X138Q/R, X140Y, X144A/E/R, X145E/H, X148ENT, X150P, X151G/RN, X153F/I, X154G/R, X155G/M/R/T/W, X156M, X157Q/V, X158D/N, X160T, X161P/Y, X162K, X163L, X164V, X166L/M, X167H, N174A, X176G/I/M, X177D/E/L/R/T, X178F/L, X179D/S/W, X180C/R, X181D/E/L/V, X182G/I/K/R, X185G/I/P, X186H, X187P, X188D/E/I/R/S/W, X189L/N, X190I/K/L, X191V/W, X192A, X193C/L/V, X195F/H/I/N/W, X196D, X197F/P, X198S, X200F, X205K, X206C, X207L/M, X208R, X209N/T/Y, X211H/L/N/R, X212F, X215E/Y, X216G/Q, X218P/Q/R, X220A/H, X221D/K, X224R, X225C/M, X226M, X227G, X228H, X231A, X235E, X236I, X238G, X239C, X240Q/R/T, X241F, X242E, X244A/P/R, X245H, X246A/P/V, X247N, X253P/V, X258P, X263N, X264R, X266A/T, X267H, X268N, X270L, X273F, X275V, X277A, X278C, X280I, X281S/Y, X283A/E/F/M/T, X284C/L/Q, X285D, X286Y, X288D/H/Q, X303G, X306T/W, X3081, X310L/V, X313Q, X316L, X318F/L/M, X331V, X337G, X338E, X339P, X341R, X351C, X352G, X355F/L/S/W, X359E, X361C/F/L, X362L, X363H, X365N, X368S, X370A/I, X373W, X374Y, X376K/P/R, X377H/K, X380H/R, X382H/Q, X384S, X3871/L, X388L, X3891/M/L/V, X393A, X396G, X397L, X398Y, X3991, X400S, X401A/C/L/S/TN, X402V, X404I/L, X405A/C/F/G/UV, X406Y, X408L, X409TN/W/Y, X410D/H/R, X411R, X412V, X413L/R, X414K, X416L/V, X417V, X418I/N/R/V, X419S, X421D/G/I/L/P/R/S/V, X424M, X426R/T, X429E/K/R, X430A/H/I, X432C/Q, X433H/K/N/R/Y, X436A, X4421, X443T, X446F, X452E/G/N, X458E/N/Q, X463V, X465K, X466G/Q, X474L/R, X478E, X482R, X487R/Y, X489F, X490C/S, X491M, X494Y, X495C/S, X496D/G, X498G/N, X499R/S/V, X500H/N, X501C/F/W, X502A/Q/R/S/W, X503C/K, X504D/E/S/T, X505E/G/K, X506G/L/M/R/W, X507H/Q, X508D/G/H/L/M, X509D/G/H/P/Q/R, X510D/E/L/M/N/R/S, X511D/G/I/K/P/R/S/T, and/or X512M/R, wherein the position is numbered with reference to a wild-type M. algicola FAR (e.g., SEQ ID NO:2), and wherein the FAR variant, when expressed in a host cell, exhibits increased fatty alcohol production of C12 to C14 or C12 to C16 fatty alcohols relative to the wild-type FAR homolog from which the FAR variant is derived. In some embodiments, the FAR variant produces an increased amount of fatty alcohols as compared to the wild-type FAR. In some embodiments, the FAR variant produces an increased amount of an aggregate comprising one or more of the fatty alcohols C12:0 (1-dodecanol), C12:1 (cis A⁵-dodecenol), C14:0 (1-tetradecanol), C14:1 (cis Δ⁷-1-tetradecanol), C16:1 (cis Δ⁹-1-hexadecenol), and C16:0 (1-hexadecanol), as compared to the wild-type FAR. In some embodiments, the FAR variant produces a fatty alcohol profile having a decreased amount of C18:0 (1-octadecanol) and/or a decreased amount of C18:1 (cis Δ¹¹-1-octadecenol) as compared to the wild-type FAR. In some embodiments, the FAR variant produces a fatty alcohol profile having an increased amount of C12:0 (1-dodecanol), C12:1 (cis Δ⁵-dodecenol), C14:0 (1-tetradecanol), and/or C14:1 (cis Δ⁷-1-tetradecanol). In some embodiments, the fatty alcohol profile produced by the FAR variant comprises an increased amount of C12:0 (1-dodecanol) as compared to the wild-type FAR. In some embodiments, the fatty alcohol profile produced by the FAR variant further comprises an increased amount of C12:1 (cis Δ⁵-dodecenol) as compared to the wild-type FAR. In some embodiments, the fatty alcohol profile produced by the FAR variant further comprises an increased amount of C14:0 (1-tetradecanol) as compared to the wild-type FAR.

In some embodiments, the present invention relates to a method of making FAR variants having increased fatty alcohol production and/or an improved fatty alcohol profile relative to wild-type FAR. In some embodiments, the method comprises:

-   -   (a) identifying a sequence that comprises at least about 70% (or         at least about 75%, at least about 80%, at least about 85%, at         least about 90%, at least about 95%, at least about 96%, at         least about 97%, at least about 98%, or at least about 99%)         sequence identity to SEQ ID NO:2 (alternatively, SEQ ID NO:4);     -   (b) aligning the identified sequence with the sequence of SEQ ID         NO:2 (alternatively, SEQ ID NO:4); and     -   (c) substituting one or more amino acid residues from the         identified sequence, wherein the substitutions are made at one         or more positions corresponding to positions selected from 1, 2,         3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 23,         40, 43, 44, 45, 47, 49, 50, 52, 61, 62, 63, 65, 66, 68, 69, 70,         71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 83, 84, 85, 86, 87,         88, 89, 90, 91, 93, 97, 98, 100, 101, 103, 104, 106, 107, 108.         111, 112, 113, 115, 116, 118, 120, 121, 122, 123, 126, 128, 129,         130, 131, 132, 133, 134, 135, 136, 137, 138, 140, 144, 145, 148,         150, 151, 153, 154, 155, 156, 157, 158, 160, 161, 162, 163, 164,         166, 167, 174, 176, 177, 178, 179, 180, 181, 182, 185, 186, 187,         188, 189, 190, 191, 192, 193, 195, 196, 197, 198, 200, 205, 206,         207, 208, 209, 211, 212, 215, 216, 218, 220, 221, 224, 225, 226,         227, 228, 231, 235, 236, 238, 239, 240, 241, 242, 244, 245, 246,         247, 253, 258, 263, 264, 266, 267, 268, 270, 273, 275, 277, 278,         280, 281, 283, 284, 285, 286, 288, 303, 306, 308, 310, 313, 316,         318, 331, 337, 338, 339, 341, 351, 352, 355, 359, 361, 362, 363,         365, 368, 370, 373, 374, 376, 377, 380, 382, 384, 387, 388, 389,         393, 396, 397, 398, 399, 400, 401, 402, 404, 405, 406, 308, 409,         410, 411, 412, 413, 414, 416, 417, 418, 419, 421, 424, 426, 429,         430, 432, 433, 436, 442, 443, 446, 452, 458, 463, 465, 466, 474,         478, 482, 487, 489, 490, 491, 494, 495, 496, 498, 499, 500, 501,         502, 503, 504, 505, 506, 507, 508, 509, 510, 511, or 512 when         the positions are aligned with SEQ ID NO:2.

In some embodiments, step (c) of the method comprises making one or more amino acid substitutions selected from M1E/G/UR/V/W, A2D/F/G/H/P/Q/R/S/T/W/Y, T3/I/L, Q4/N/R/S/W/Y, Q5M/N, Q6C/H/K/P/R/S/V/Y, Q7H/N, N8A/E/H/V, G9C/F/V, A10T, S11D/G, A12D/R/S/T, S13G/L/V, G14K/L/M/R/V, V15I, L16G/I/S, E17C/G/H/R, Q181/L, R20K, H23R, K40R, R43H, T44A, V45A/S, D47E/N, G49E, G50A, H52Y, H61R, P62Q, A63D, R65G, E66D/F/S/V, F68A/V, L69E/I/M/Q, N70D/E/L/M/R/T, E71C/M/Q/S, 172L, A73G/H/K/L/M, S74K/L/P/T/W, S75C/E/H/N, S76E/F/I/L/R, V77I/P/T, F78M, E79D/I/L/Q/V, R80I/L, L81F/T, H83E, D84A, D85E, N86S, E87V, A88G/V, F89D/N/P/R, E90D/Q, T91A/R, L93D, V97I, H98P, I100V, T101A, E103C/S/V, V104I/M, E106A/H, S107A, R108E/G/H/Q, L111I, T112G, P113I/Q, R115D, F116Y, A118K, A120C, G121T, Q122E/H/T, V123L, F126V, N128C/H/L, S129D, A130C/S, A131P/S, S132H, V133A/G, N134D/K/R/S/V, F135E, R136L, E137L, E138Q/R, D140Y, K144A/E/R, 1145E/H, L148E/K/T, L150P, E151G/R/V, V153F/I, A154G/R, A155G/M/R/T/W, L156M, A157Q/V, E158D/N, N160T, S161P/Y, A162K, M163L, A164V, I166L/M, Q167H, N174A, K176G/I/M, N177D/E/L/R/T, S178F/L, G179D/S/W, Q180C/R, I181D/E/L/V, T182G/I/K/R, V185G/I/P, I186H, K187P, P188D/E/I/R/S/W, A189L/N, G190I/K/L, E191V/W, S192A, I193C/L/V, R195F/H/I/N/W, S196D, T197F/P, D198S, Y200F, E205K, L206C, V207L/M, H208R, L209N/T/Y, Q211H/L/N/R, D212F, S215EN, D216G/Q, K218P/Q/R, R220A/H, Y221D/K, K224R, V225C/M, L226M, E227G, K228H, V231A, 1235E, R236I, A238G, N239C, N240Q/R/T, Y241F, G242E, S244A/P/R, D245H, T246A/P/V, Y247N, L253P/V, L258P, S263N, G264R, S266A/T, L267H, T268N, V270L, S273F, 1275V, S277A, A278C, E280I, E281S/Y, S283A/E/F/M/T, P284C/L/Q, G285D, W286Y, E288D/H/Q, E303G, S306T/W, F3081, G310L/V, S313Q, I316L, V318F/L/M, S331V, S337G, G338E, S339P, Q341R, G351C, S352G, I355F/L/S/W, K359E, I361C/F/L, D362L, Y363H, M365N, A368S, T370A/I, A373W, A374Y, D376K/P/R, Q377H/K, Y380H/R, R382H/Q, T384S, F3871/L, V388L, A3891/M/L/V, K393A, D396G, V397L, V398Y, V399I, G400S, G401A/C/L/S/TN, M402V, V404I/L, P405A/C/F/G/L/V, L406Y, I408L, A409T/V/W/Y, G410D/H/R, K411R, A412V, M413L/R, R414K, A416L/V, G417V, Q418I/N/RN, N419S, E421D/G/I/L/P/R/S/V, V424M, K426R/T, D429E/K/R, T430A/H/I, R432C/Q, S433H/K/N/R/Y, T436A, T4421, A443T, Y446F, S452E/G/N, S458E/N/Q, L463V, R465K, V466G/Q, Q474L/R, Q478E, C482R, G487R/Y, L489F, N490C/S, R491M, L494Y, K495C/S, E496D/G, K498G/N, L499R/S/V, Y500H/N, S501C/F/W, L502A/Q/R/S/W, R503c/K, A504D/E/S/T, A505E/G/K, D506G/L/M/R/W, T507H/Q, R508D/G/H/L/M, K509D/G/H/P/Q/R, K510D/E/L/M/N/R/S, A511D/G/I/K/P/R/S/T, and/or A512M/R.

In some embodiments, the method further comprises determining whether the one or more amino acid substitutions increase fatty alcohol production and/or an improved fatty alcohol profile in comparison to the wild-type FAR from which the FAR variant is derived.

M. aquaeolei FAR Variants

In a related aspect, FAR variants derived from M. aquaeolei FAR (SEQ ID NO:4) are provided. Table 11 shows exemplary substitutions and substitution sets. In one aspect, the invention provides a FAR variant comprising at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:4 and comprising a substitution at a substitution position (or set of positions) disclosed in Table 11. These variants are sometimes referred to as “FAR Maq variants.” It will be appreciated, as noted above, that SEQ ID NO:4 and SEQ ID NO:2 share about 78% sequence identity. Accordingly, FAR Maq variants can be viewed as a subgenus of FAR variants of the invention to which all disclosure herein is applicable.

In some embodiments, a FAR variant comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:4 and comprises a substitution relative to SEQ ID NO:4 at one or more positions selected from position 8, position 74, position 116, position 135, position 228, position 411, position 430, position 434, position 438, position 503, position 512, and position 513, wherein the position is numbered with reference to SEQ ID NO:4. In some embodiments, the amino acid residue in the FAR variant at position 8 relative to SEQ ID NO:4 is histidine (H8); the amino acid residue in the FAR variant at position 74 relative to SEQ ID NO:4 is alanine (A74); the amino acid residue in the FAR variant at position 116 relative to SEQ ID NO:4 is aspartic acid (D116); the amino acid residue in the FAR variant at position 135 relative to SEQ ID NO:4 is asparagine (N135); the amino acid residue in the FAR variant at position 228 relative to SEQ ID NO:4 is glutamic acid (E228); the amino acid residue in the FAR variant at position 411 relative to SEQ ID NO:4 is aspartic acid (D411); the amino acid residue in the FAR variant at position 430 relative to SEQ ID NO:4 is aspartic acid (D430); the amino acid residue in the FAR variant at position 434 relative to SEQ ID NO:4 is serine (S434); the amino acid residue in the FAR variant at position 438 relative to SEQ ID NO:4 is isoleucine (I438); the amino acid residue in the FAR variant at position 503 relative to SEQ ID NO:4 is leucine (L503); the amino acid residue in the FAR variant at position 512 relative to SEQ ID NO:4 is alanine (A512); and the amino acid residue in the FAR variant at position 513 relative to SEQ ID NO:4 is alanine (A513). In some embodiments, a FAR variant comprises an amino acid substitution at residue H8K that is lysine (H8K). In some embodiments, a FAR variant comprises an amino acid substitution at residue A74 that is selected from lysine and leucine (A74K/L). In some embodiments, a FAR variant comprises an amino acid substitution at residue D116 that is selected from alanine and glutamic acid (D116A/E). In some embodiments, a FAR variant comprises an amino acid substitution at residue N135 that is lysine (N135K). In some embodiments, a FAR variant comprises an amino acid substitution at residue E228 that is glycine (E228G). In some embodiments, a FAR variant comprises an amino acid substitution at residue D411 that is arginine (D411R). In some embodiments, a FAR variant comprises an amino acid substitution at residue D430 that is lysine (D430K). In some embodiments, a FAR variant comprises an amino acid substitution at residue S434 that is selected from lysine, phenylalanine, and tryptophan (S434K/F/W). In some embodiments, a FAR variant comprises an amino acid substitution at residue I438 that is valine (I438V). In some embodiments, a FAR variant comprises an amino acid substitution at residue L503 that is selected from arginine and serine (L503R/S). In some embodiments, a FAR variant comprises an amino acid substitution at residue A512 that is selected from glycine, proline, and glutamine (A512G/P/Q). In some embodiments, a FAR variant comprises an amino acid substitution at residue A513 that is selected from glycine, lysine, proline, arginine, serine, threonine (and A513G/K/P/R/S/T). In some embodiments, a FAR variant comprises 100% amino acid sequence identity to SEQ ID NO:4 except for amino acid substitutions at one or more of positions H8, A74, D116, N135, E228, D411, D430, S434, I1438, L503, A512, and A513 as numbered with reference to SEQ ID NO:4.

FAR Maq variants of the invention as described herein may have any of the improved properties disclosed hereinabove, such as increased total fatty alcohol production, increased production of fatty alcohols at at a specified culture pH or over an increased pH range, or changes in fatty alcohol profile as compared to a wild-type FAR.

FAR Maq variants of the invention may comprise a substitution or substitution set exemplified in Table 11. For example, FAR Maq Variant No. 1047 (see Table 11) has a lysine (substituted for asparagine) at position 135 (numbered with reference to SEQ ID NO:4). More broadly, FAR Maq variants of the invention may comprise a substitution at a position or set of positions exemplified in Table 11. As a non-limiting example, FAR Maq Variant No. 1047 (see Table 11) has a substitution for asparagine at position 135 (wherein the positions are numbered with reference to SEQ ID NO:4), and accordingly a particular FAR Maq variant of the invention may comprise a substitution at position 135 (i.e., any residue other than asparagine), and optionally any other substitutions as described herein, e.g., as described in Table 11.

FAR Motifs

In some embodiments, a FAR variant of the invention comprises an amino acid motif that is conserved among FAR enyzmes such as bacterial FARs and/or plant FARs. The characteristics of FARs from plants have been described, e.g., in Rowland and Domergue, Plant Sci. 193-194:28-38 (2012). Plant FAR proteins are about 500 amino acids long, and some contain an amino terminal extension of ˜50 to 120 amino acids that serves as a transit peptide to target the protein to the organelles. In certain embodiments, a FAR enzyme or functional fragment thereof that is especially suitable for the production of fatty alcohols is identified by the presence of one or more domains, which are found in proteins with FAR activity. In various embodiments, the one or more domains are identified by multiple sequence alignments using hidden Markov models (“HMMs”) to search large collections of protein families, for example, the Pfam collection available at pfam.sanger.ac.uk/. See R. D. Finn et al. (2008) Nucl. Acids Res. Database issue 36:D281-D288.

In certain embodiments, the one or more protein domains by which FAR enzymes are identified is a NAD binding domain. In some embodiments, the NAD binding domain is categorized in the “NAD_binding_(—)4” domain family (Pfam domain PF07993). The NAD_binding_(—)4 domain is characterized by the motif [I,V,F]-X-[I,L,V]-T-G-X-T-G-F-L-[G,A]. See Aarts et al., Plant J. 12:615-623 (1997), incorporated by reference herein. This motif is believed to be involved in NAD(P)H binding. In some embodiments, the NAD binding domain is near the N-terminus. As shown in FIG. 2, inspection of bacterial FARs closely related to M. algicola FAR (Oceanobacter RED65, M. aquaeolei, marine bacteria HP15, and Hahella KCTC 2396) revealed the presence of the TGxxGxxG motif. In certain embodiments, the one or more protein domains by which FAR enzymes are identified is a “sterile” domain (Pfam domain PF03015). In some embodiments, the sterile domain is near the C-terminus. In certain embodiments, a FAR variant of the invention comprises a NAD binding domain near the N-terminus and a sterile domain near the C-terminus.

Additionally, FAR enzymes from Marinobacter and other bacteria contain the classic YxxxK active site motif of the short-chain dehydrogenase/reductase superfamily (Kavanaugh et al., Cell. Mol. Life. Sci. 65:3895-3906 (2008)). Inspection of bacterial FARs closely related to M. algicola FAR (Oceanobacter RED65, M. aquaeolei, marine bacteria HP15, and Hahella KCTC 2396) revealed the presence of a YxxxK motif, as shown in FIG. 2. Furthermore, as shown in FIG. 3, an inspection of an alignment between the YxxxK motif region from M. algicola FAR and that of some FARs from plants, insects, or mouse revealed that this motif can be extended to YxxxKxxxE. Further comparisons showed that in another family of bacterial FARs, represented by the FAR from M. aquaeolei VT8 (protein YP_(—)959769), the motif YxxxK could be extended to YxxxKxxxK/R. It is predicted that changing any one or more of these three conserved residues in the motif (for example, by changing to alanine) will drastically affect enzymatic activity.

In some embodiments, a FAR enzyme of the invention is identified by the presence of the TGxxGxxG motif near the N-terminus and/or the presence of the YxxxKxxxE/K/R motif. In one embodiment, the present invention relates to FAR variants comprising at least about 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with a wild-type FAR polypeptide and comprising one or more mutations (e.g. amino acid substitutions) as compared to the wild-type FAR from which the FAR variant is derived, wherein the FAR variant comprises the amino acid motif TGxxGxxG. In another embodiment, the present invention relates to FAR variants comprising at least about 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with a wild-type FAR polypeptide and comprising one or more mutations (e.g. amino acid substitutions) as compared to the wild-type FAR from which the FAR variant is derived, wherein the FAR variant comprises the amino acid motif YxxxKxxxE/K/R. In some embodiments, the FAR variant comprises both the TGxxGxxG motif and the YxxxKxxxE/K/R motif.

Additional Metabolic Engineering

In one embodiment, the recombinant host cell exhibiting increased production of shorter chain fatty alcohols (e.g., increased amount of shorter chain fatty alcohols and/or an increased proportion of shorter chain fatty alcohols in a fatty alcohol composition) further contains an exogenous gene operably linked to a promoter that is functional in the microbial organism. The incorporation of the exogenous gene can be accomplished as described herein or using any techniques well known in the art.

In some embodiments, the host cell can be modified to express or over-express one or more genes encoding enzymes, other than FAR, that are involved in fatty acyl-CoA derivative biosynthesis. In particular embodiments, the gene encodes a fatty acid biosynthetic (“Fab”) enzyme, an acyl-CoA synthetase (“ACS”), or an acyl-ACP thioesterase (“TE”). In some embodiments, one or more Fab enzymes are overexpressed in a host cell. See, e.g., U.S. Provisional Application No. 61/577,756, filed Dec. 20, 2011, incorporated by reference herein. There are at least 8 enzymes involved fatty acid initiation and elongation biosynthesis, including FabA, FabB, FabD, FabF, FabG, FabH, Fabl, and FabZ. In some embodiments, the gene encodes “FabH,” β-ketoacyl-ACP synthase III (EC 2.3.1.180). In some embodiments, the host cell comprises a polynucleotide sequence encoding an E. coli FabH protein. Polynucleotides encoding FabH enzymes are known in the art. See, e.g., Tsay et al., 1992, J. Biol. Chem. 267:6807-6814. In some embodiments, the gene encodes “Fabl,” a trans-2-enoyl-ACP reductase (EC 1.3.1.9 and 1.3.1.10). In some embodiments, the gene encodes “FabZ,” a beta-hydroxyacyl-ACP dehydratase (EC 4.2.1.59 to 4.2.61). In some embodiments, the gene encodes “FadD,” an acyl-CoA synthetase (EC 6.2.1). In some embodiments, the gene encodes TE, a thioesterase (EC 3.1.2.1 to EC 3.1.2.27 and also EC3.1.1.5 and EC 3.1.2.-). When multiple exogenous genes are expressed, in some embodiments, the expression vector encoding a first enzyme (e.g., a FAR variant) and the expression vector encoding a second enzyme (e.g., a Fab enzyme, ACS, or TE) are separate nucleic acids. In other embodiments, the first enzyme and the second enzyme are encoded on the same expression vector, and expression of each enzyme is independently regulated by a different promoter.

V. Polynucleotides and Expression Systems for Expressing FAR Variants

In another aspect, the present invention provides polynucleotides encoding the FAR variants as described herein. In some embodiments, the invention relates to a polynucleotide sequence that encodes an improved FAR polypeptide wherein the polynucleotide encodes a fatty acyl reductase (FAR) polypeptide comprising a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, or SEQ ID NO:28; and said improved FAR polypeptide comprising one or more substitutions (e.g., one or more of the specified substitutions or substitution sets as described herein, e.g., one or more substitutions or substitution sets in any of Tables 1, 2, 6, 7, 8, 9, 10, or 11).

The polynucleotide can be a DNA or RNA, and can be single-stranded or double-stranded. The polynucleotide may be operably linked to one or more heterologous regulatory or control sequences that control gene expression to create a recombinant polynucleotide capable of expressing the polypeptide. Expression constructs containing a heterologous polynucleotide encoding the FAR variant can be introduced into appropriate host cells to express the FAR variant.

In some embodiments, the FAR variant is generated from a wild-type FAR polynucleotide sequence (e.g., a wild-type M. algicola FAR polynucleotide sequence of SEQ ID NO:1 or a wild-type M. aquaeolei FAR polynucleotide sequence of SEQ ID NO:3) or the portion thereof comprising the open reading frame, with changes made as required at the codons corresponding to substitutions (residues mutated relative to the wild-type sequence as described herein, for example at any of Table 1, Table 2, Table 4, Table 7, Table 8, Table 9, Table 10, or Table 11). In addition, one or more “silent” nucleotide changes can be incorporated. In certain embodiments, the FAR variant is generated from a reference sequence that is a variant FAR polynucleotide sequence (e.g., the variant encoded by the sequence of any of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:27 or the portion thereof comprising the open reading frame, with changes made as required at the codons corresponding to substitutions. In addition, one or more “silent” nucleotide changes can be incorporated.

The availability of a polypeptide sequence of a specific FAR variant polypeptide provides a description of all polynucleotides capable of encoding that enzyme or fragment because of the known correspondence between amino acids and the genetic code. For most organisms the genetic code is “Amino Acid (one letter code) [codons]”: phenylalanine (F) [TTT, TTC]; leucine (L) [TTA, TTG, CTT, CTC, CTA, CTG]; isoleucine (I) [ATT, ATC, ATA]; methionine (M) [ATG]; valine (V) [TGG, GTC, GTA, GTG]; serine (S) [TCT, TCC, TCA, TCG, AGT, AGC]; proline (P) [CCT, CCC, CCA, CCG]; threonine (T) [ACT, ACC, ACA, ACG]; alanine (A) [GCT, GCC, GCA, GCG]; tyrosine (Y) [TAT, TAC]; histidine (H) [CAT, CAC]; glutamine (Q) [CAA, CAG]; asparagine (N) [AAT, AAC]; lysine (K) AAA, AAG]; aspartic acid (D) [GAT, GAC]; glutamic acid (E) [GAA, GAG]; cysteine (C) [TGT, TGC]; tryptophan (W) [TGG]; arginine (R) [CGT, CGC, CGA, CGG, AGA, AGG]; and glycine (G) [GGT, GGC, GGA, GGG]. In certain embodiments, the degeneracy of the genetic code is used to produce a large number of polynucleotides that encode the variant FAR polypeptides described herein. In some embodiments, the polynucleotides that encode the variant FAR polypeptides described herein are codon optimized for expression in specific microorganisms. In particular embodiments, the polynucleotides that encode the variant FAR polypeptides described herein are codon optimized for expression in bacteria, yeast (such as S. cerevisiae or Y. lipolytica) or filamentous fungi. In other specific embodiments, the polynucleotides are codon optimized for expression in E. coli. Codon schemes and/or methods for determining codon schemes optimized for particular microorganisms of interest are well known (see, e.g., the references cited with the definition of “preferred, optimal high, codon usage bias codons”; see also http://www.kazusa.or.jp/codon/).

A variety of methods are known for determining the codon frequency (e.g., codon usage, relative synonymous codon usage) and codon preference in specific organisms, including multivariate analysis, for example, using cluster analysis or correspondence analysis, and the effective number of codons used in a gene (see GCG CodonPreference, Genetics Computer Group Wisconsin Package; Codon W, John Peden, University of Nottingham; McInerney, J. O, 1998, Bioinformatics 14:372-73; Stenico et al., 1994, Nucleic Acids Res. 222437-46; Wright, F., 1990, Gene 87:23-29; Wada et al., 1992, Nucleic Acids Res. 20:2111-2118; Nakamura et al., 2000, Nucl. Acids Res. 28:292; Henaut and Danchin, “Escherichia coli and Salmonella,” 1996, Neidhardt, et al. Eds., ASM Press, Washington D.C., p. 2047-2066, all of which are incorporated herein by reference). The data source for obtaining codon usage may rely on any available nucleotide sequence capable of coding for a protein. These data sets include nucleic acid sequences actually known to encode expressed proteins (e.g., complete protein coding sequences-CDS), expressed sequence tags (ESTs), or predicted coding regions of genomic sequences (see for example, Mount, D., Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Uberbacher, E. C., 1996, Methods Enzymol. 266:259-281; Tiwari et al., 1997, Comput. Appl. Biosci. 13:263-270, all of which are incorporated herein by reference).

In some embodiments, the present invention provides a method for making a FAR polynucleotide variant, wherein the method comprises (1) introducing one or more mutations into a polynucleotide encoding a FAR which comprises at least 70% (alternatively, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% and even 100%) sequence identity to the amino acid sequence of SEQ ID NO:2 or a functional fragment thereof to produce a modified polynucleotide and wherein the polynucleotide encodes a FAR variant having at least 80% (alternatively, at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:2 and comprises a substitution at one or more positions selected from positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 23, 40, 43, 44, 45, 47, 49, 50, 52, 61, 62, 63, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 83, 84, 85, 86, 87, 88, 89, 90, 91, 93, 97, 98, 100, 101, 103, 104, 106, 107, 108. 111, 112, 113, 115, 116, 118, 120, 121, 122, 123, 126, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 140, 144, 145, 148, 150, 151, 153, 154, 155, 156, 157, 158, 160, 161, 162, 163, 164, 166, 167, 174, 176, 177, 178, 179, 180, 181, 182, 185, 186, 187, 188, 189, 190, 191, 192, 193, 195, 196, 197, 198, 200, 205, 206, 207, 208, 209, 211, 212, 215, 216, 218, 220, 221, 224, 225, 226, 227, 228, 231, 235, 236, 238, 239, 240, 241, 242, 244, 245, 246, 247, 253, 258, 263, 264, 266, 267, 268, 270, 273, 275, 277, 278, 280, 281, 283, 284, 285, 286, 288, 303, 306, 308, 310, 313, 316, 318, 331, 337, 338, 339, 341, 351, 352, 355, 359, 361, 362, 363, 365, 368, 370, 373, 374, 376, 377, 380, 382, 384, 387, 388, 389, 393, 396, 397, 398, 399, 400, 401, 402, 404, 405, 406, 308, 409, 410, 411, 412, 413, 414, 416, 417, 418, 419, 421, 424, 426, 429, 430, 432, 433, 436, 442, 443, 446, 452, 458, 463, 465, 466, 474, 478, 482, 487, 489, 490, 491, 494, 495, 496, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, or 512, wherein the position is numbered with reference to SEQ ID NO:2; transforming a host cell with the modified polynucleotide; and screening the transformed host cell for an improvement in a desired phenotype relative to a corresponding transformed host cell comprising a polynucleotide encoding a reference FAR such as a wild-type FAR comprising at least 70% (alternatively, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100%) sequence identity to the amino acid sequence of SEQ ID NO:2 or a functional fragment thereof. In some embodiments, the modified polynucleotide sequence encodes a FAR variant having one or more substitutions selected from M1E/G/L/R/V/W, A2D/F/G/H/P/Q/R/S/T/W/Y, T3/I/L, Q4/N/R/S/W/Y, Q5M/N, Q6C/H/K/P/R/S/V/Y, Q7H/N, N8A/E/H/V, G9C/F/V, A10T, S11D/G, A12D/R/S/T, S13G/L/V, G14K/L/M/R/V, V151, L16G/I/S, E17C/G/H/R, Q181/L, R20K, H23R, K40R, R43H, T44A, V45A/S, D47E/N, G49E, G50A, H52Y, H61R, P62Q, A63D, R65G, E66D/F/S/Y, F68A/V, L69E/I/M/Q, N70D/E/L/M/R/T, E71C/M/Q/S, 172L, A73G/H/K/L/M, S74K/L/P/T/W, S75C/E/H/N, S76E/F/I/L/R, V77I/P/T, F78M, E79D/I/L/Q/V, R80I/L, L81F/T, H83E, D84A, D85E, N86S, E87V, A88G/V, F89D/N/P/R, E90D/Q, T91A/R, L93D, V97I, H98P, 1100V, T101A, E103C/S/V, V104I/M, E106A/H, S107A, R108E/G/H/Q, L111I, T112G, P113I/Q, R115D, F116Y, A118K, A120C, G121T, Q122E/H/T, V123L, F126V, N128C/H/L, S129D, A130C/S, A131P/S, S132H, V133A/G, N134D/K/R/S/Y, F135E, R136L, E137L, E138Q/R, D140Y, K144A/E/R, I145E/H, L148E/K/T, L150P, E151G/RN, V153F/I, A154G/R, A155G/M/R/T/W, L156M, A157Q/V, E158D/N, N160T, S161P/Y, A162K, M163L, A164V, I166L/M, Q167H, N174A, K176G/I/M, N177D/E/L/R/T, S178F/L, G179D/S/W, Q180C/R, 1181D/E/L/V, T182G/I/K/R, V185G/I/P, I186H, K187P, P188D/E/I/R/S/W, A189L/N, G1901/K/L, E191V/W, S192A, I193C/L/V, R195F/H/I/N/W, S196D, T197F/P, D198S, Y200F, E205K, L206C, V207L/M, H208R, L209N/T/Y, Q211H/L/N/R, D212F, S215E/Y, D216G/Q, K218P/Q/R, R220A/H, Y221D/K, K224R, V225C/M, L226M, E227G, K228H, V231A, I235E, R2361, A238G, N239C, N240Q/R/T, Y241F, G242E, S244A/P/R, D245H, T246A/P/V, Y247N, L253P/V, L258P, S263N, G264R, S266A/T, L267H, T268N, V270L, S273F, 1275V, S277A, A278C, E2801, E281S/Y, S283A/E/F/M/T, P284C/L/Q, G285D, W286Y, E288D/H/Q, E303G, S306T/W, F3081, G310L/V, S313Q, I316L, V318F/L/M, S331V, S337G, G338E, S339P, Q341R, G351C, S352G, I355F/L/S/W, K359E, 1361C/F/L, D362L, Y363H, M365N, A368S, T370A/I, A373W, A374Y, D376K/P/R, Q377H/K, Y380H/R, R382H/Q, T384S, F387I/L, V388L, A389I/M/L/V, K393A, D396G, V397L, V398Y, V399I, G400S, G401A/C/L/S/TN, M402V, V404I/L, P405A/C/F/G/L/V, L406Y, 1408L, A409T/V/W/Y, G410D/H/R, K411R, A412V, M413L/R, R414K, A416L/V, G417V, Q418I/N/R/V, N419S, E421D/G/I/L/P/R/S/V, V424M, K426R/T, D429E/K/R, T430A/H/I, R432C/Q, S433H/K/N/R/Y, T436A, T4421, A443T, Y446F, S452E/G/N, S458E/N/Q, L463V, R465K, V466G/Q, Q474L/R, Q478E, C482R, G487R/Y, L489F, N490C/S, R491M, L494Y, K495C/S, E496D/G, K498G/N, L499R/S/V, Y500H/N, S501C/F/W, L502A/Q/R/S/W, R503c/K, A504D/E/S/T, A505E/G/K, D506G/L/M/R/W, T507H/Q, R508D/G/H/L/M, K509D/G/H/P/Q/R, K510D/E/L/M/N/R/S, A511D/G/I/K/P/R/S/T and/or A512M/R. In some embodiments the modified polynucleotide sequence encoding a FAR variant will comprise at least 85% (alternatively, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% at least 98%, or at least 99%) sequence identity with nucleic acid sequence of SEQ ID NO:1.

In some embodiments, the present invention provides a method for making a FAR polynucleotide variant, wherein the method comprises introducing one or more mutations into a polynucleotide encoding a FAR which comprises at least 90% (alternatively, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100%) sequence identity to the amino acid sequence of any of SEQ ID NOs:6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or 28, or a functional fragment thereof, to produce a modified polynucleotide, wherein the modification is selected from the group consisting of a substitution, a deletion, and an insertion; transforming a host cell with the modified polynucleotide; and screening the transformed host cell for an improvement in a desired phenotype relative to a corresponding transformed host cell comprising a polynucleotide encoding the variant FAR having at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% and even 100%) sequence identity to the amino acid sequence of the FAR polypeptide from which the FAR variant was derived. Exemplary desired phenotypes include improved fatty alcohol production, improved total and/or secreted fatty alcohol composition, and/or alteration of the fatty alcohol composition (including, but not limited to, an increase in the amount of C12 to C14 fatty alcohols produced, or a profile comprising, for example, one or more, an increased amount of C12:0 (1-dodecanol), an increased amount of C14:0 (1-tetradecanol), a decreased amount of C16:0 (1-hexadecanol), and a decreased amount of C18:1 (cis Δ¹¹-1-octadecenol) as compared to a wild-type FAR (e.g., SEQ ID NO:2 or SEQ ID NO:4) or a reference FAR (e.g., any of SEQ ID NOs:6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or 28). In some embodiments, the modified polynucleotide sequence encoding a FAR variant will comprise at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100%) sequence identity with nucleic acid sequence of any of SEQ ID NOs:5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27.

In some embodiments, the FAR variant comprises an amino acid sequence encoded by a nucleic acid that hybridizes under moderate, stringent or highly stringent conditions over substantially the entire length of a nucleic acid corresponding to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:27.

Certain silent mutations have also been identified in polynucleotides encoding the variant FAR polypeptides which appear to confer the property of increased fatty alcohol production in transformed E. coli cells as compared to wild-type M. algicola DG893 FAR (see Table 1). The silent mutations include: t6a, t6g, t6c, t9c, t9g, t27c, a30g, a51g, a81t, a147t, c171t, t174c, t180c, t226a, a237g, g243a, a318g, c321g, c321a, a336c, t339g, t363c, c402t, t459c, a474g, a540g, t564g, a615g, g627t, t628c, a633g, g681a, g711a, t792c, t834c, t870c, t927c, t967c, c994t, t1026c, t1149c, c1173t, t1203c, t1236g, t1248c, g1263a, g1272a, c1281t, t1287c, g1290c, t1297a, c1299g, t1326c, t1357c, c1366t, t1372a, t1374g, t1398c, t1410c, t1413c, t1435c, t1461g, g1485a, g1497t, t1501a, t1504c, t1515g, t1515a, t1521c, t1524c, a1527g, and t1533c (where nucleotide position is determined by alignment with SEQ ID NO:1).

Polynucleotide Synthesis

Polynucleotides encoding variant FAR polypeptides can be prepared using methods that are well known in the art. Typically, oligonucleotides of up to about 40 bases are individually synthesized, then joined (e.g., by enzymatic or chemical ligation methods, or polymerase-mediated methods) to form essentially any desired continuous sequence. For example, polynucleotides of the present invention can be prepared by chemical synthesis using, for example, the classical phosphoramidite method described by Beaucage, et al., 1981, Tetrahedron Letters, 22:1859-69, or the method described by Matthes, et al., 1984, EMBO J. 3:801-05, both of which are incorporated herein by reference. These methods are typically practiced in automated synthetic methods. According to the phosphoramidite method, oligonucleotides are synthesized, e.g., in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors.

In addition, essentially any nucleic acid can be custom ordered from any of a variety of commercial sources, such as The Midland Certified Reagent Company (Midland, Tex.), The Great American Gene Company (Ramona, Calif.), ExpressGen Inc. (Chicago, Ill.), Operon Technologies Inc. (Alameda, Calif.), and many others.

Polynucleotides may also be synthesized by well-known techniques as described in the technical literature. See, e.g., Carruthers, et al., 1982, Cold Spring Harbor Symp. Quant. Biol., 47:411-18 and Adams et al., 1983, J. Am. Chem. Soc. 105:661, both of which are incorporated herein by reference. Double stranded DNA fragments may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.

General texts that describe molecular biological techniques which are useful herein, including the use of vectors, promoters, protocols sufficient to direct persons of skill through in vitro amplification methods, including the polymerase chain reaction (PCR) and the ligase chain reaction (LCR), and many other relevant methods, include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al., Molecular Cloning—A Laboratory Manual (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989 (“Sambrook”) and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 2009) (“Ausubel”), all of which are incorporated herein by reference. Reference is made to Berger, Sambrook, and Ausubel, as well as Mullis et al., (1987) U.S. Pat. No. 4,683,202; PCR Protocols A Guide to Methods and Applications (Innis et al. eds) Academic Press Inc. San Diego, Calif. (1990) (Innis); Arnheim & Levinson (Oct. 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991) 3, 81-94; (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86, 1173; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lomeli et al. (1989) J. Clin. Chem. 35, 1826; Landegren et al., (1988) Science 241, 1077-1080; Van Brunt (1990) Biotechnology 8, 291-294; Wu and Wallace, (1989) Gene 4, 560; Barringer et al. (1990) Gene 89, 117, and Sooknanan and Malek (1995) Biotechnology 13: 563-564, all of which are incorporated herein by reference. Methods for cloning in vitro amplified nucleic acids are described in Wallace et al., U.S. Pat. No. 5,426,039, which is incorporated herein by reference.

Vectors

The present invention further provides DNA constructs and vectors comprising polynucleotides encoding the variant FAR polypeptides for expression in heterologous recombinant host cells. In certain embodiments, the DNA constructs and vectors comprise a polynucleotide sequence that encodes any one of the variant FAR polypeptides as disclosed above. In certain embodiments, the DNA constructs and vectors comprise a polynucleotide sequence as encompassed by the invention and disclosed herein above. In certain embodiments, the DNA constructs and vectors comprise a polynucleotide sequence that encodes a variant FAR polypeptide herein, wherein the variant FAR is a full-length FAR. In other embodiments, the DNA constructs and vectors comprise a polynucleotide sequence that encodes a variant FAR polypeptide, wherein the variant FAR is a functional fragment of a variant full-length FAR enzyme. In certain embodiments, the polynucleotides encoding variant FAR polypeptides for expression in heterologous recombinant host cells as described herein are operably linked to a promoter, and optionally, to other control sequences.

In a particular aspect the present invention provides an expression vector comprising a FAR polynucleotide operably linked to a heterologous promoter. Expression vectors of the present invention may be used to transform an appropriate host cell to permit the host to express the FAR protein. Methods for recombinant expression of proteins in bacteria, yeast, and other organisms are well known in the art, and a number expression vectors are available or can be constructed using routine methods.

A recombinant expression vector can be any vector, e.g., a plasmid or a virus, which can be manipulated by recombinant DNA techniques to facilitate expression of a variant FAR polypeptide in a recombinant host cell. In certain embodiments, the expression vectors is stably integrated into the chromosome of the recombinant host cell and comprises one or more heterologous genes operably linked to one or more control sequences useful for production of a variant FAR polypeptide. In other embodiments, the expression vector is an extrachromosomal replicative DNA molecule, e.g., a linear or closed circular plasmid, that is found either in low copy number (e.g., from about 1 to about 10 copies per genome equivalent) or in high copy number (e.g., more than about 10 copies per genome equivalent). Expression vectors which, in certain embodiments, are useful for expressing variant FAR enzymes as disclosed herein are commercially available, e.g., from Sigma-Aldrich Chemicals, St. Louis, Mo. and Stratagene, LaJolla, Calif. In some embodiments, examples of suitable expression vectors are plasmids which are derived from pBR322 (Gibco BRL), pUC (Gibco BRL), pREP4, pCEP4 (Invitrogen) or pPoly (Lathe et al., 1987, Gene 57:193-201).

In certain embodiments, the present disclosure provides a plasmid for expression of heterologous genes in E. coli. Expression vector pCK110900, which comprises a P15A origin of replication “ori” (P15A ori), lac a CAP binding site, a lac promoter, a T7 ribosomal binding site (T7g10 RBS) and a chloramphenicol resistance gene (camR). This expression vector is depicted in FIG. 3 of U.S. Patent Publication No. 2006/0195947, which is incorporated herein by reference in its entirety. Other suitable plasmid vectors include derivatives of pCL1920 and pCL1921 (Lerner and Inouye, 1990; NAR 18:4631). These vectors contain the pSC101 ori and confer resistance to spectinomycin (GenBank:AB236930). In some embodiments the vector is an expression vector derived from pCL1920 including the Trc promoter and the lacIq gene from E. coli. In some embodiments the vector is pLS8379 or an expression vector derived from pLS8379. In some embodiments the vector is pCDX11 or an expression vector derived from pCDX11.

In certain embodiments, the present disclosure provides a replicating plasmid for expression of heterologous genes in Yarrowia, and particularly in Y. lipolytica.

In various embodiments, an expression vector optionally contains a ribosome binding site (RBS) for translation initiation, and a transcription terminator, such as PinII. RBS are effective control elements for protein production and the type of RBS can result in different expression levels of the same protein. One skilled in the art is aware of a number of well-characterized RBS. See, e.g., Vellanoweth and Rabinowitz, 1992, Mol. Microbiol. 6: 1105-1114. In some embodiments, the RBS is from the native E. coli FabA gene (5′ ATAAAATAAGGCTTACAGAGAA; SEQ ID NO:47) or from the native E. coli FabH gene (5′ ACCGAAAAGTGACTGAGCGTAC; SEQ ID NO:48). The vector also optionally includes appropriate sequences for amplifying expression, e.g., an enhancer.

Promoters

Suitable promoters include constitutive promoters, regulated promoters, and inducible promoters. Appropriate promoter sequences can be obtained from genes encoding extracellular or intracellular polypeptides which are either endogenous or heterologous to the host cell. Methods for the isolation, identification and manipulation of promoters of varying strengths are available in or readily adapted from the art. See, e.g., Nevoigt et al. (2006) Appl. Environ. Microbiol. 72:5266-5273, the disclosure of which is herein incorporated by reference in its entirety.

In certain embodiments, the DNA constructs and vectors comprising a polynucleotide encoding a variant FAR polypeptides are suitable for expression in bacteria. For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the present disclosure, include the promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, Bacillus megaterium promoters, and prokaryotic beta-lactamase gene (VIIIa-Kamaroff et al., Proc. Natl. Acad. Sci. USA 75: 3727-3731 (1978)), as well as the tac promoter (DeBoer et al., Proc. Natl. Acad. Sci. USA 80: 21-25 (1993)). Additional promoters include trp promoter, phage lambda PL, T7 promoter, promoters found at PromEC and the like. Promoters suitable for use in the present disclosure are described in Terpe H., 2006, Appl. Microbiol. Biotechnol. 72:211-222 and in Sambrook et al (2001) Molecular Cloning: A Laboratory Manual, 3^(rd) ed., Cold Spring Harbor Laboratory Press, New York.

In various embodiments, the DNA constructs and vectors comprising polynucleotides encoding a variant FAR polypeptide are suitable for expression in yeast. In certain embodiments, the DNA constructs and vectors comprising the polynucleotides encoding a variant FAR are suitable for expression in oleaginous yeast, such as but not limited to Yarrow lipolytica. In certain embodiments the promoter is a Y. lipolytica promoter.

In certain embodiments, the DNA constructs and vectors comprising the polynucleotides encoding a variant FAR polypeptide are suitable for expression in yeast, such as but not limited to S. cerevisiae. For yeast host cells, suitable promoters for directing transcription of the nucleic acid constructs of the present disclosure are known to the skilled artisan and include, but are not limited to, an enolase (ENO-1_gene) promoter, a galactokinase (GAL1) promoter, an alcohol dehyrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP) promoter, a translation elongation factor EF-1 alpha (TEF1) promoter as well as those described by Romanos et al. (1992) Yeast 8:423-488. In other embodiments, promoters include the TEF1 promoter and an RPS7 promoter.

In various embodiments, the DNA constructs and vectors comprising polynucleotides encoding a variant FAR polypeptide are suitable for expression in filamentous fungal host cells. For these cells, suitable promoters for directing the transcription of the nucleic acid constructs of the present disclosure include promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (WO 96/00787), as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase), and mutant, truncated, and hybrid promoters thereof. Examples of suitable promoters useful for directing the transcription of the nucleotide constructs of the present invention in a filamentous fungal host cell are promoters such as cbh1, cbh2, egl1, egl2, pepA, hfb1, hfb2, xyn1, amy, and glaA (Nunberg et al., Mol. Cell. Biol., 4:2306-2315 (1984), Boel et al., EMBO J. 3:1581-1585 ((1984) and EPA 137280).

Other Regulatory Elements

In various embodiments, the polynucleotides useful for expressing heterologous FAR enzymes in recombinant host cells are operably linked to other control sequences, including but not limited to, a transcription terminator sequence, a signal sequence that when translated directs the expressed polypeptide into the secretory pathway of the recombinant host cell, and a polyadenylation sequence (eukaryotes). The choice of appropriate control sequences for use in the polynucleotide constructs of the present disclosure is within the skill in the art and in various embodiments is dependent on the recombinant host cell used and the desired method of recovering the fatty alcohol compositions produced.

In various embodiments, the expression vector includes one or more selectable markers, which permit easy selection of transformed cells. Selectable markers for use in a host organism as described herein include, but are not limited to, genes that confer antibiotic resistance (e.g., ampicillin, kanamycin, chloramphenicol or tetracycline resistance) to the recombinant host organism that comprises the vector.

VI. Recombinant Host Cells Expressing Far Variants and Producing Fatty Alcohols

In some embodiments, the present invention provides a method for producing a recombinant host cell, wherein the method comprises: (a) providing a nucleic acid construct of the present invention, wherein the nucleic acid construct comprises polynucleotide encoding a FAR variant polypeptide as described herein; and (b) transforming a host cell with the nucleic acid construct to produce a recombinant cell wherein the FAR variant is produced. In certain embodiments, the present invention provides a recombinant microorganism (e.g., a bacterial microorganism, e.g., Ecol) engineered to produce a fatty alcohol composition comprising a polynucleotide sequence encoding a FAR variant polypeptide, wherein the FAR variant is any one of the FAR variants described herein (e.g., a FAR variant comprising one or more substitution sets listed in any of Table 1, Table 2, Table 4, Table 7, Table 8, Table 9, Table 10, or Table 11).

In some embodiments, the host cell is a bacterial cell. In some embodiments, the host cell is a yeast cell. The engineered host cell is cultured in a suitable nutrient medium under conditions permitting the expression of the variant FAR enzyme. The medium used to culture the cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g., in catalogues of the American Type Culture Collection).

Host Cells

The recombinant host cells of the present invention generally comprise a polynucleotide, such as one of the polynucleotides described above, encoding an improved FAR polypeptide. Suitable host cells include, but are not limited to, bacteria, yeast, filamentous fungi, and algae. In certain embodiments, the host cell is a bacteria, e.g., E. coli. Cells which are useful in the practice of the present disclosure are readily accessible from a number of culture collections and other sources, e.g., the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ) (German Collection of Microorganisms and Cell Culture), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

In certain embodiments, the engineered microorganisms according to the invention are wild-type microorganisms. In other embodiments, the engineered microorganisms are genetically modified microorganisms. As used herein, “genetically modified” microorganisms include microorganisms having one or more endogenous genes removed, microorganisms having one or more endogenous genes with reduced expression compared to the parent or wild-type microorganism, or microorganisms having one or more genes overexpressed compared to the parent or wild-type microorganism. In certain embodiments, the one or more genes that are overexpressed are endogenous to the microorganism. In some embodiments, the one or more genes that are overexpressed are heterologous to the microorganism.

Prokaryotic Host Cells

In some embodiments, the host cell is a prokaryotic cell. Suitable prokaryotic cells include gram positive, gram negative and gram-variable bacterial cells. In certain embodiments, host cells include, but are not limited to, species of a genus selected from the group consisting of Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Acinetobacter, Acidothermus, Arthrobacter, Azobacter, Bacillus, Bifidobacterium, Brevibacterium, Butyrivibrio, Buchnera, Campestris, Camplyobacter, Clostridium, Corynebacterium, Chromatium, Coprococcus, Cyanobacteria, Escherichia, Enterococcus, Enterobacter, Erwinia, Fusobacterium, Faecalibacterium, Francisella, Flavobacterium, Geobacillus, Haemophilus, Helicobacter, Klebsiella, Lactobacillus, Lactococcus, Ilyobacter, Micrococcus, Microbacterium, Mesorhizobium, Methylobacterium, Methylobacterium, Mycobacterium, Neisseria, Pantoea, Pseudomonas, Prochlorococcus, Rhodobacter, Rhodopseudomonas, Rhodopseudomonas, Roseburia, Rhodospirillum, Rhodococcus, Scenedesmun, Streptomyces, Streptococcus, Synnecoccus, Saccharomonospora, Staphylococcus, Serratia, Salmonella, Shigella, Thermoanaerobacterium, Tropheryma, Tularensis, Temecula, Thermosynechococcus, Thermococcus, Ureaplasma, Xanthomonas, Xylella, Yersinia and Zymomonas. In particular embodiments, the host cell is a species of a genus selected from the group consisting of Agrobacterium, Arthrobacter, Bacillus, Clostridium, Corynebacterium, Escherichia, Erwinia, Geobacillus, Klebsiella, Lactobacillus, Mycobacterium, Pantoea, Rhodococcus, Streptomyces and Zymomonas.

In certain embodiments, the recombinant host cell is an industrial bacterial strain. Numerous bacterial industrial strains are known and suitable for use in the methods disclosed herein. In some embodiments, the bacterial host cell is a species of the genus Bacillus, e.g., B. thuringiensis, B. megaterium, B. subtilis, B. lentus, B. circulans, B. pumilus, B. lautus, B. coagulans, B. brevis, B. pumilus, B. licheniformis, B. alkaophius, B. licheniformis, B. clausii, B. stearothermophilus, B. halodurans, and B. amyloliquefaciens. In some embodiments the bacterial host cell is a species of the genus Erwinia, e.g., E. uredovora, E. carotovora, E. ananas, E. herbicola, E. punctata or E. terreus. In other embodiments the bacterial host cell is a species of the genus Pantoea, e.g., P. citrea or P. agglomerans. In still other embodiments, the bacterial host cell is a species of the genus Streptomyces, e.g., S. ambofaciens, S. achromogenes, S. avermitilis, S. coelicolor, S. aureofaciens, S. aureus, S. fungicidicus, S. griseus or S. lividans. In further embodiments, the bacterial host cell is a species of the genus Zymomonas, e.g., Z. mobilis or Z. lipolytica. In further embodiments, the bacterial host cell is a species of the genus Rhodococcus, e.g. R. opacus.

In particular embodiments, the bacterial host cell is a species of the genus Escherichia, e.g., E. coli. In certain embodiments, the E. coli is a wild-type bacterium. In various embodiments, the wild-type E. coli bacterial strain useful in the processes described herein is selected from, but not limited to, strain W3110, strain MG1655 and strain BW25113. In other embodiments, the E. coli is genetically modified. Examples of genetically modified E. coli useful as recombinant host cells include, but are not limited to, genetically modified E. coli found in the Keio Collection, available from the National BioResource Project at NBRP E. coli, Microbial Genetics Laboratory, National Institute of Genetics 1111 Yata, Mishima, Shizuoka, 411-8540.

Yeast Host Cells

In certain embodiments, the recombinant host cell is a yeast. In various embodiments, the yeast host cell is a species of a genus selected from the group consisting of Candida, Hansenula, Saccharomyces, Schizosaccharomyces, Rhototorua, Issatchenkia, Rhodosporidium, Pichia, Kluyveromyces, and Yarrowia. In particular embodiments, the yeast host cell is a species of a genus selected from the group consisting of Saccharomyces, Candida, Pichia and Yarrowia.

In various embodiments, the yeast host cell is selected from the group of Rhodotorula (e.g., R. glutinous, R. graminis, R. mucilaginosa, R. minuta, R. bacarum), Rhodosporidium toruloides, Hansenula polymorpha, Saccharomyces (e.g., S. cerevisiae, S. carlsbergensis, S. diastaticus, S. norbensis, and S. kluyveri,). Schizosaccharomyces pombe, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia ferniemtans, Issatchenkia orientalis, Pichia kodamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia quercuum, Pichia pijperi, Pichia stipitis, Pichia methanolica, Pichia angusta, Kluyveromyces lactis, Candida (e.g., C. revkaufi, C. pulcherrima, C. tropicalis, C. utills, C. curvata D, C. curvata R, C. diddensiae, C. boldinii, C. albicans, C. krusei, C. ethanolic), Yarrowia (e.g. Y. paralipolytica, and Y. lipolytica), Cryptococcus (terricolus) albidus var. albidus, Cryptococcus laurentii, Trichosporon pullans, Trichosporon cutaneum, Trichosporon cutancum, Trichosporon pullulans, Lipomyces starkeyii, Lipomyces lipoferus, Lipomyces tetrasporus, Endomycopsis vernalis, Hansenula ciferri, Hansenula saturnus, and Trigonopsis variables and synonyms or taxonomic equivalents thereof. In certain embodiments, the yeast is Y. lipolytica. In certain embodiments, Yarrowia lipolytica strains include, but are not limited to, DSMZ 1345, DSMZ 3286, DSMZ 8218, DSMZ 70561, DSMZ 70562, DSMZ 21175, ATCC 20362, ATCC 18944 and ATCC 76982. In certain embodiments, the yeast host cell is a wild-type cell. In various embodiments, the wild-type yeast cell strain is selected from, but not limited to, strain BY4741, strain FL100a, strain INVSC1, strain NRRL Y-390, strain NRRL Y-1438, strain NRRL YB-1952, strain NRRL Y-5997, strain NRRL Y-7567, strain NRRL Y-1532, strain NRRL YB-4149 and strain NRRL Y-567. In other embodiments, the yeast host cell is genetically modified. Examples of genetically modified yeast useful as recombinant host cells include, but are not limited to, genetically modified yeast found in the Open Biosystems collection found at http://www.openbiosystems.com/GeneExpression/Yeast/YKO/. See Winzeler et al. (1999) Science 285:901-906.

In yet other embodiments, the recombinant host cell is a filamentous fungus. In certain embodiments, the filamentous fungal host cell is a species of a genus selected from the group consisting of Achlya, Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Cephalosporium, Chrysosporium, Cochliobolus, Corynascus, Ctyphonectria, Cryptococcus, Coprinus, Coriolus, Diplodia, Endothis, Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea, Myceliophthora, Mucor, Neurospora, Penicillium, Podospora, Phlebia, Piromyces, Pyricularia, Rhizomucor, Rhizopus, Schizophyllum, Scytalidium, Sporotrichum, Talaromyces, Thermoascus, Thielavia, Trametes, Tolypocladium, Trichoderma, Verticillium, Volvariella, and teleomorphs, synonyms or taxonomic equivalents thereof.

In some embodiments, the filamentous fungal host cell is an Aspergillus species, a Chrysosporium species, a Corynascus species, a Fusarium species, a Humicola species, a Myceliophthora species, a Neurospora species, a Penicillum species, a Tolypocladium species, a Tramates species, or Trichoderma species. In other embodiments, the Trichoderma species is selected from T. longibrachiatum, T. viride, Hypocrea jecorina and T. reesei; the Aspergillus species is selected from A. awamori, A. funigatus, A. japonicus, A. nidulans, A. niger, A. aculeatus, A. foetidus, A. oryzae, A. sojae, and A. kawachi; the Chrysosporium species is C. lucknowense; the Fusarium species is selected from F. graminum, F. oxysporum and F. venenatum; the Myceliophthora species is M. thermophilia; the Neurospora species is N. crassa; the Humicola species is selected from H. insolens, H. grisea, and H. lanuginosa; the Penicillum species is selected from P. purpurogenum, P. chrysogenum, and P. verruculosum; the Thielavia species is T. terrestris; and the Trametes species is selected from T. villosa and T. versicolor.

In some embodiments, the filamentous fungal host is a wild-type organism. In other embodiments, the filamentous fungal host is genetically modified.

Transformation and Cell Culture

In some embodiments, a host cell is transformed with a polynucleotide encoding a FAR variant as described herein. In transformation, the polynucleotide that is introduced into the host cell remains in the genome or on a plasmid or other stably maintained vector in the cell and is capable of being inherited by the progeny thereof. Stable transformation is typically accomplished by transforming the host cell with an expression vector comprising the polynucleotide of interest (e.g., the polynucleotide encoding the FAR variant) along with a selectable marker gene (e.g., a gene that confers resistance to an antibiotic). Only those host cells which have integrated the polynucleotide sequences of the expression vector into their genome will survive selection with the marker (e.g., antibiotic). These stably transformed host cells can then be propagated according to known methods in the art.

Methods, reagents and tools for transforming host cells described herein, such as bacteria, yeast (including oleaginous yeast) and filamentous fungi are known in the art. General methods, reagents and tools for transforming, e.g., bacteria can be found, for example, in Sambrook et al (2001) Molecular Cloning: A Laboratory Manual, 3^(rd) ed., Cold Spring Harbor Laboratory Press, New York. Methods, reagents and tools for transforming yeast are described in “Guide to Yeast Genetics and Molecular Biology,” C. Guthrie and G. Fink, Eds., Methods in Enzymology 350 (Academic Press, San Diego, 2002). Methods, reagents and tools for transforming, culturing, and manipulating Y. lipolytica are found in “Yarrowia lipolytica,” C. Madzak, J. M. Nicaud and C. Gaillardin in “Production of Recombinant Proteins. Novel Microbial and Eucaryotic Expression Systems,” G. Gellissen, Ed. 2005, which is incorporated herein by reference for all purposes. In some embodiments, introduction of the DNA construct or vector of the present invention into a host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, PEG-mediated transformation, electroporation, or other common techniques (See Davis et al., 1986, Basic Methods in Molecular Biology, which is incorporated herein by reference).

The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying the FAR polynucleotide. Culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to those skilled in the art. As noted, many references are available for the culture and production of many cells, including cells of bacterial, plant, animal (especially mammalian) and archebacterial origin. See e.g., Sambrook, Ausubel, and Berger (all supra), as well as Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, third edition, Wiley-Liss, New York and the references cited therein; Doyle and Griffiths (1997) Mammalian Cell Culture: Essential Techniques John Wiley and Sons, NY; Humason (1979) Animal Tissue Techniques, fourth edition W.H. Freeman and Company; and Ricciardelli, et al., (1989) In Vitro Cell Dev. Biol. 25:1016-1024, all of which are incorporated herein by reference. For plant cell culture and regeneration, Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, N.Y.; Gamborg and Phillips (eds) (1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg New York); Jones, ed. (1984) Plant Gene Transfer and Expression Protocols, Humana Press, Totowa, N. J. and Plant Molecular Biology (1993) R. R. D. Croy, Ed. Bios Scientific Publishers, Oxford, U.K. ISBN 0 12 198370 6, all of which are incorporated herein by reference. Cell culture media in general are set forth in Atlas and Parks (eds.) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla., which is incorporated herein by reference. Additional information for cell culture is found in available commercial literature such as the Life Science Research Cell Culture Catalogue (1998) from Sigma-Aldrich, Inc (St Louis, Mo.) (“Sigma-LSRCCC”) and, for example, The Plant Culture Catalogue and supplement (1997) also from Sigma-Aldrich, Inc (St Louis, Mo.) (“Sigma-PCCS”), all of which are incorporated herein by reference.

VII. Methods of Producing Fatty Alcohols

The present disclosure also provides methods of producing fatty alcohols with the FAR variant polypeptides described herein, as well as the resultant fatty alcohol compositions produced by said methods.

In some particular embodiments, a method of producing a fatty alcohol composition comprises culturing a recombinant microorganism in a suitable culture medium, wherein the recombinant microorganism comprises a gene encoding a FAR variant polypeptide capable of producing at least about 1.5 more fatty alcohols than a wild-type FAR comprising SEQ ID NO:2 when assayed under the same conditions, or as compared to a reference FAR variant (e.g., a reference FAR variant comprising SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:14) when assayed under the same conditions.

In some embodiments, a method of producing a fatty alcohol composition comprises culturing a recombinant microorganism in a suitable culture medium, wherein the recombinant microorganism comprises a gene encoding a FAR variant capable of producing a fatty alcohol profile having one or more of an increased amount of C12:0 (1-dodecanol), an increased amount of C12:1 (cis Δ⁵-dodecenol), an increased amount of C14:0 (1-tetradecanol), an increased amount of C14:1 (cis Δ⁷-1-tetradecanol), a decreased amount of C16:0 (1-hexadecanol), a decreased amount of C16:1 (cis Δ⁹-1-hexadecenol), a decreased amount of C18:0 (1-octadecanol), or a decreased amount of C18:1 (cis Δ¹¹-1-octadecenol) as compared to a wild-type FAR comprising SEQ ID NO:2 or SEQ ID NO:4. In some embodiments, a method of producing a fatty alcohol composition comprises culturing a recombinant microorganism in a suitable culture medium, wherein the recombinant microorganism comprises a gene encoding a FAR variant capable of producing an increased amount of C10 to C18 fatty alcohols, an increased amount of C12 to C16 fatty alcohols, an increased amount of C12 to C14 fatty alcohols, an increased amount of C14 to C16 fatty alcohols, or a decreased amount of C18 fatty alcohols as compared to a wild-type FAR comprising SEQ ID NO:2 when assayed under the same conditions, or as compared to a reference FAR variant (e.g., a reference FAR variant comprising SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:14) when assayed under the same conditions.

In some embodiments, the method comprises culturing a recombinant microorganism (for example, but not limited to a strain of E. coli) in a suitable culture medium, wherein the recombinant microorganism comprises a gene encoding a FAR variant polypeptide comprising a sequence that is at least about 70% (or at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%) identical to SEQ ID NO:2 or SEQ ID NO:4 and comprises one or more amino acid substitutions selected from M1E/G/L/R/V/W, A2D/F/G/H/P/Q/R/S/T/W/Y, T3/I/L, Q4/N/R/S/W/Y, Q5M/N, Q6C/H/K/P/R/S/V/Y, Q7H/N, N8A/E/HN, G9C/F/V, A10T, S11D/G, A12D/R/S/T, S13G/L/V, G14K/L/M/RN, V151, L16G/I/S, E17C/G/H/R, Q181/L, R20K, H23R, K40R, R43H, T44A, V45A/S, D47E/N, G49E, G50A, H52Y, H61R, P62Q, A63D, R65G, E66D/F/S/Y, F68AN, L69E/I/M/Q, N70D/E/L/M/R/T, E71C/M/Q/S, 172L, A73G/H/K/L/M, S74K/L/P/T/W, S75C/E/H/N, S76E/F/I/L/R, V771/P/T, F78M, E79D/I/UQN, R80I/L, L81F/T, H83E, D84A, D85E, N86S, E87V, A88G/V, F89D/N/P/R, E90D/Q, T91A/R, L93D, V971, H98P, I100V, T101A, E103C/S/V, V104I/M, E106A/H, S107A, R108E/G/H/Q, L111I, T112G, P113I/Q, R115D, F116Y, A118K, A120C, G121T, Q122E/H/T, V123L, F126V, N128C/H/L, S129D, A130C/S, A131P/S, S132H, V133A/G, N134D/K/R/S/Y, F135E, R136L, E137L, E138Q/R, D140Y, K144A/E/R, 1145E/H, L148E/K/T, L150P, E151G/RN, V153F/I, A154G/R, A155G/M/R/T/W, L156M, A157QN, E158D/N, N160T, S161P/Y, A162K, M163L, A164V, 1166L/M, Q167H, N174A, K176G/1/M, N177D/E/L/R/T, S178F/L, G179D/S/W, Q180C/R, 1181D/E/L/V, T182G/I/K/R, V185G/I/P, I186H, K187P, P188D/E/I/R/S/W, A189L/N, G190I/K/L, E191V/W, S192A, I193C/L/V, R195F/H/I/N/W, S196D, T197F/P, D198S, Y200F, E205K, L206C, V207L/M, H208R, L209N/T/Y, Q211H/L/N/R, D212F, S215E/Y, D216G/Q, K218P/Q/R, R220A/H, Y221D/K, K224R, V225C/M, L226M, E227G, K228H, V231A, I235E, R2361, A238G, N239C, N240Q/R/T, Y241F, G242E, S244A/P/R, D245H, T246A/P/V, Y247N, L253P/V, L258P, S263N, G264R, S266A/T, L267H, T268N, V270L, S273F, 1275V, S277A, A278C, E2801, E281S/Y, S283A/E/F/M/T, P284C/L/Q, G285D, W286Y, E288D/H/Q, E303G, S306T/W, F3081, G310UV, S313Q, I316L, V318F/L/M, S331V, S337G, G338E, S339P, Q341R, G351C, S352G, I355F/L/S/W, K359E, I361C/F/L, D362L, Y363H, M365N, A368S, T370A/I, A373W, A374Y, D376K/P/R, Q377H/K, Y380H/R, R382H/Q, T384S, F3871/L, V388L, A389I/M/L/V, K393A, D396G, V397L, V398Y, V3991, G400S, G401A/C/L/S/TN, M402V, V4041/L, P405A/C/F/G/L/V, L406Y, 1408L, A409TN/W/Y, G410D/H/R, K411R, A412V, M413L/R, R414K, A416UV, G417V, Q418I/N/R/V, N419S, E421D/G/I/L/P/R/S/V, V424M, K426R/T, D429E/K/R, T430A/H/I, R432C/Q, S433H/K/N/R/Y, T436A, T4421, A443T, Y446F, S452E/G/N, S458E/N/Q, L463V, R465K, V466G/Q, Q474L/R, Q478E, C482R, G487R/Y, L489F, N490C/S, R491M, L494Y, K495C/S, E496D/G, K498G/N, L499R/S/V, Y500H/N, S501C/F/W, L502A/Q/R/S/W, R503C/K, A504D/E/S/T, A505E/G/K, D506G/L/M/R/W, T507H/Q, R508D/G/H/L/M, K509D/G/H/P/Q/R, K510D/E/L/M/N/R/S, A511D/G/I/K/P/R/S/T, and/or A512M/R, wherein the amino acid positions are numbered with reference to SEQ ID NO:2; and allowing expression of said gene, wherein said expression results in the production of a composition of fatty alcohols (e.g., a composition having an increased amount of C12:0 (1-dodecanol), an increased amount of C12:1 (cis Δ⁵-dodecenol), an increased amount of C14:0 (1-tetradecanol), an increased amount of C14:1 (cis Δ⁷-1-tetradecanol), an increased amount of C16:0 (1-hexadecanol), an increased amount of C16:1 (cis Δ⁹-1-hexadecenol), a decreased amount of C18:0 (1-octadecanol), or a decreased amount of C18:1 (cis Δ¹¹-1-octadecenol)); or a composition comprising a fatty alcohol profile having one or more of an increased amount of C12:0 (1-dodecanol), an increased amount of C12:1 (cis Δ⁵-dodecenol), an increased amount of C14:0 (1-tetradecanol), an increased amount of C14:1 (cis Δ⁷-1-tetradecanol), an increased amount of C16:0 (1-hexadecanol), an increased amount of C16:1 (cis Δ⁹-1-hexadecenol), a decreased amount of C18:0 (1-octadecanol), or a decreased amount of C18:1 (cis Δ¹¹-1-octadecenol)) as compared to a wild-type FAR).

While not meant to limit the invention in any manner, in some embodiments, the method of producing a fatty alcohol composition comprises:

-   -   a) culturing a recombinant strain of E. coli, Yarrowia, or         Saccharomyces, in a suitable culture medium, wherein the         recombinant strain comprises a gene encoding a FAR variant         polypeptide,     -   b) allowing expression of said gene, and     -   c) producing the fatty alcohol composition, wherein i) the FAR         variant polypeptide comprises an amino acid sequence that is at         least about 70% identical to SEQ ID NO:2 or SEQ ID NO:4 and         comprises one or more amino acid substitutions as described         herein (e.g., one or more amino acid substitutions sets listed         in Table 1, Table 2, Table 4, Table 7, Table 8, Table 9, Table         10, or Table 11); ii) the culturing is carried at a temperature         of about 20° C. to about 40° C. and from about 16 to 120         hours, iii) the culture medium comprises a carbon source         comprising fermentable sugars obtained from a cellulosic         feedstock, and iv) at least about 5 g/L of recoverable fatty         alcohols are produced. In some embodiments, fermentable sugars         in the culture medium include glucose and/or sucrose.

In some embodiments, the method of producing a fatty alcohol composition comprises:

-   -   a) culturing a recombinant strain of E. coli a suitable culture         medium, wherein the recombinant strain comprises a gene encoding         a FAR variant polypeptide,     -   b) allowing expression of said gene, and     -   c) producing the fatty alcohol composition, wherein i) the FAR         variant polypeptide comprises comprises an amino acid sequence         having at least 90% (at least 91%, at least 92%, at least 93%,         at least 94%, at least 95%, at least 96%, at least 97%, at least         98%, or at least 99%) sequence identity to any of SEQ ID NOs: 2,         4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or 28, and         comprises a substitution at one or more positions selected from         1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,         20, 23, 40, 43, 44, 45, 47, 49, 50, 52, 61, 62, 63, 65, 66, 68,         69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 83, 84, 85,         86, 87, 88, 89, 90, 91, 93, 97, 98, 100, 101, 103, 104, 106,         107, 108. 111, 112, 113, 115, 116, 118, 120, 121, 122, 123, 126,         128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 140, 144,         145, 148, 150, 151, 153, 154, 155, 156, 157, 158, 160, 161, 162,         163, 164, 166, 167, 174, 176, 177, 178, 179, 180, 181, 182, 185,         186, 187, 188, 189, 190, 191, 192, 193, 195, 196, 197, 198, 200,         205, 206, 207, 208, 209, 211, 212, 215, 216, 218, 220, 221, 224,         225, 226, 227, 228, 231, 235, 236, 238, 239, 240, 241, 242, 244,         245, 246, 247, 253, 258, 263, 264, 266, 267, 268, 270, 273, 275,         277, 278, 280, 281, 283, 284, 285, 286, 288, 303, 306, 308, 310,         313, 316, 318, 331, 337, 338, 339, 341, 351, 352, 355, 359, 361,         362, 363, 365, 368, 370, 373, 374, 376, 377, 380, 382, 384, 387,         388, 389, 393, 396, 397, 398, 399, 400, 401, 402, 404, 405, 406,         308, 409, 410, 411, 412, 413, 414, 416, 417, 418, 419, 421, 424,         426, 429, 430, 432, 433, 436, 442, 443, 446, 452, 458, 463, 465,         466, 474, 478, 482, 487, 489, 490, 491, 494, 495, 496, 498, 499,         500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, or         512, wherein the position is numbered with reference to SEQ ID         NO:2; ii) the culturing is carried at a temperature of about         20° C. to about 40° C. and from about 16 to 120 hours; iii) the         culture medium comprises a carbon source comprising fermentable         sugars; and iv) at least about 5 g/L of fatty alcohol are         produced. In certain embodiments, one or more substitutions are         selected from M1E/G/URN/W, A2D/F/G/H/P/Q/R/S/T/W/Y, T3/I/L,         Q4/N/R/S/W/Y, Q5M/N, Q6C/H/K/P/R/S/V/Y, Q7H/N, N8A/E/H/V,         G9C/FN, A10T, 311D/G, A12D/R/S/T, S13G/L/V, G14K/L/M/RN, V15I,         L16G/I/S, E17C/G/H/R, Q181/L, R20K, H23R, K40R, R43H, T44A,         V45A/S, D47E/N, G49E, G50A, H52Y, H61R, P62Q, A63D, R65G,         E66D/F/S/Y, F68AN, L69E/I/M/Q, N70D/E/L/M/R/T, E71C/M/Q/S, I72L,         A73G/H/K/L/M, S74K/L/P/T/W, S75C/E/H/N, S76E/F/I/L/R, V77I/P/T,         F78M, E79D/I/L/Q/V, R80I/L, L81F/T, H83E, D84A, D85E, N86S,         E87V, A88G/V, F89D/N/P/R, E90D/Q, T91A/R, L93D, V971, H98P,         I100V, T101A, E103C/S/V, V104I/M, E106A/H, S107A, R108E/G/H/Q,         L111I, T112G, P113I/Q, R115D, F116Y, A118K, A120C, G121T,         Q122E/H/T, V123L, F126V, N128C/H/L, S129D, A130C/S, A131P/S,         S132H, V133A/G, N134D/K/R/S/V, F135E, R136L, E137L, E138Q/R,         D140Y, K144A/E/R, I145E/H, L148E/K/T, L150P, E151G/R/V, V153F/I,         A154G/R, A155G/M/R/T/W, L156M, A157Q/V, E158D/N, N160T, S161P/Y,         A162K, M163L, A164V, 1166L/M, Q167H, N174A, K176G/I/M,         N177D/E/L/R/T, S178F/L, G179D/S/W, Q180C/R, I181D/E/L/V,         T182G/I/K/R, V185G/I/P, I186H, K187P, P188D/E/I/R/S/W, A189L/N,         G190I/K/L, E191V/W, S192A, I193C/L/V, R195F/H/I/N/W, S196D,         T197F/P, D198S, Y200F, E205K, L206C, V207L/M, H208R, L209N/T/Y,         Q211H/L/N/R, D212F, S215E/Y, D216G/Q, K218P/Q/R, R220A/H,         Y221D/K, K224R, V225C/M, L226M, E227G, K228H, V231A, 1235E,         R236I, A238G, N239C, N240Q/R/T, Y241F, G242E, S244A/P/R, D245H,         T246A/P/V, Y247N, L253P/V, L258P, S263N, G264R, S266A/T, L267H,         T268N, V270L, S273F, 1275V, S277A, A278C, E2801, E281S/Y,         S283A/E/F/M/T, P284C/L/Q, G285D, W286Y, E288D/H/Q, E303G,         S306T/W, F3081, G310L/V, S313Q, I316L, V318F/L/M, S331V, S337G,         G338E, S339P, Q341R, G351C, S352G, I355F/L/S/W, K359E,         I361C/F/L, D362L, Y363H, M365N, A368S, T370A/I, A373W, A374Y,         D376K/P/R, Q377H/K, Y380H/R, R382H/Q, T384S, F3871/L, V388L,         A389I/M/L/V, K393A, D396G, V397L, V398Y, V399I, G400S,         G401A/C/L/S/T/V, M402V, V404I/L, P405A/C/F/G/L/V, L406Y, I408L,         A409T/V/W/Y, G410D/H/R, K411R, A412V, M413L/R, R414K, A416L/V,         G417V, Q418I/N/RN, N419S, E421D/G/I/L/N/P/R/S/V, V424M, K426R/T,         D429E/K/R, T430A/H/I, R432C/Q, S433H/K/N/RN, T436A, T442I,         A443T, Y446F, S452E/G/N, S458E/N/Q, L463V, R465K, V466G/Q,         Q474L/R, Q478E, C482R, G487R/Y, L489F, N490C/S, R491M, L494Y,         K495C/S, E496D/G, K498G/N, L499R/S/V, Y500H/N, S501C/F/W,         L502A/Q/R/S/W, R503c/K, A504D/E/S/T, A505E/G/K, D506G/L/M/R/W,         T507H/Q, R508D/G/H/L/M, K509D/G/H/P/Q/R, K510D/E/L/M/N/R/S,         A511D/G/I/K/P/R/S/T, and/or A512M/R.

Fatty alcohol compositions can be made by culturing a host cell comprising a FAR variant as described herein in a suitable culture medium under conditions (e.g., time, temperature, and/or pH conditions) suitable for the production of fatty alcohols, and producing the fatty alcohol composition. In some embodiments, the methods further comprise isolating the fatty alcohol compositions from the culture medium. In some embodiments, the host cell comprising the FAR variant is cultured under temperature conditions of from about 10° C. to about 60° C. (e.g., from about 15° C. to about 50° C., from about 20° C. to about 45° C., from about 20° C. to about 40° C., from about 20° C. to about 35° C., or from about 25° C. to about 45° C.). In some embodiments, the host cell comprising the FAR variant is cultured under time conditions the fermentation of from about 8 hours to 240 hours (e.g., from about 8 hours to about 168 hours, from about 8 hours to 144 hours, from about 16 hours to about 120 hours, or from about 24 hours to about 72 hours). In some embodiments, the host cell comprising the FAR variant is cultured under pH conditions of about pH 4-8 (e.g., about pH 4.5 to 7.5, about pH 5 to 7, or about pH 5.5 to 6.5).

The methods of producing fatty alcohol compositions as described herein can be carried out in cell-free systems with isolated FAR variant polypeptides, or in cell-based systems with microorganisms engineered to express one or more FAR variant polypeptides as described herein.

In embodiments in which fatty alcohols are produced in cell-free systems, an isolated FAR variant polypeptide is provided with a substrate (a fatty acyl-ACP and/or a fatty acyl-CoA complex) and NAD(P)H under suitable conditions of temperature, pH, and ionic strength and time sufficient for the production of a fatty alcohol composition. In some embodiments, the FAR variant polypeptide is provided with a composition of a fatty acid, Coenzyme A and a fatty acyl-CoA synthase under suitable conditions of temperature, pH and ionic strength and time sufficient for production of a fatty alcohol composition.

In embodiments employing cell-based systems, a recombinant host cell capable of expressing a gene that encodes a FAR variant polypeptide as described herein above is cultured in an aqueous nutrient medium comprising an assimilable source of carbon under conditions suitable for production of a fatty alcohol composition. Any of the various host microorganisms described herein can be used.

Fermentation Methods

Fermentation of the recombinant host cell is carried out under suitable conditions and for a time sufficient for production of fatty alcohols. Conditions for the culture and production of cells, including filamentous fungi, bacterial and yeast cells, are readily available. Cell culture media in general are set forth in Atlas and Parks, Eds., The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla., which is incorporated herein by reference. The individual components of such media are available from commercial sources, e.g., under the Difco™ and BBL™ trademarks. In one non-limiting example, the aqueous nutrient medium is a “rich medium” comprising complex sources of nitrogen, salts, and carbon, such as YP medium, comprising 10 g/L of peptone and 10 g/L yeast extract of such a medium. In other non-limiting embodiments, the aqueous nutrient medium comprises a mixture of Yeast Nitrogen Base (Difco™) in combination supplemented with an appropriate mixture of amino acids, e.g., SC medium. In particular aspects of this embodiment, the amino acid mixture lacks one or more amino acids, thereby imposing selective pressure for maintenance of an expression vector within the recombinant host cell.

The recombinant microorganisms can be grown under batch or continuous fermentation conditions. Classical batch fermentation is a closed system, wherein the compositions of the medium is set at the beginning of the fermentation and is not subject to artificial alternations during the fermentation. A variation of the batch system is a fed-batch fermentation which also finds use in the present invention. In this variation, the substrate is added in increments as the fermentation progresses. Fed-batch systems are useful when catabolite repression is likely to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the medium. Batch and fed-batch fermentations are common and well known in the art. Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth. Continuous fermentation systems strive to maintain steady state growth conditions. Methods for modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology.

In some embodiments, fermentations are carried out a temperature within the range of from about 10° C. to about 60° C., from about 15° C. to about 50° C., from about 20° C. to about 45° C., from about 25° C. to about 45° C., from about 30° C. to about 45° C. and from about 25° C. to about 40° C.

In other embodiments, the fermentation is carried out for a period of time within the range of from about 8 hours to 240 hours, from about 8 hours to about 168 hours, from about 8 hours to 144 hours, from about 16 hours to about 120 hours, or from about 24 hours to about 72 hours. It will be understood that, in certain embodiments where thermostable host cells are used, fermentations may be carried out at higher temperatures.

In other embodiments, the fermentation is carried out at a pH in the range of 4 to 8, in the range of 4.5 to 7.5, in the range of 5 to 7, or in the range of 5.5 to 6.5.

Carbon sources useful in the aqueous fermentation medium or broth of the disclosed process in which the recombinant microorganisms are grown are those assimilable by the recombinant host strain. Assimilable carbon sources are available in many forms and include renewable carbon sources and the cellulosic and starch feedstock substrates obtained there from. Such examples include, for example, depolymerized cellulosic material, monosaccharides, disaccharides, oligosaccharides, saturated and unsaturated fatty acids, succinate, acetate and mixtures thereof. Further carbon sources include, without limitation, glucose, galactose, sucrose, xylose, fructose, glycerol, arabinose, mannose, raffinose, lactose, maltose, and mixtures thereof.

In some preferred embodiments, the assimilable carbon source is from cellulosic and/or starch feedstock derived from but not limited to, wood, wood pulp, paper pulp, grain (e.g., corn grain), corn stover, corn fiber, rice, paper and pulp processing waste, woody or herbaceous plants and residue, fruit or vegetable pulp, distillers grain, grasses, rice hulls, wheat straw, cotton, hemp, flax, sisal, corn cobs, sugar cane bagasse, sugar beets, sorghum, barley, barley straw, switch grass, wood chips, municipal solid wastes, aquatic crops, and mixtures thereof.

In some embodiments, the cellulosic feedstock useful as an assimilable carbon source has been derived from a biomass substrate that has been pretreated. The term “biomass” is broadly used herein to encompasses any living or dead biological material that contains a polysaccharide substrate, including but not limited to cellulose, starch, other forms of long-chain carbohydrate polymers, and mixtures of such sources. A biomass substrate is “pretreated,” using methods known in the art, such as chemical pretreatment (e.g., ammonia pretreatment, dilute acid pretreatment, dilute alkali pretreatment, or solvent exposure), physical pretreatment (e.g., steam explosion or irradiation), mechanical pretreatment (e.g., grinding or milling) and biological pretreatment (e.g., application of lignin-solubilizing microorganisms) and combinations thereof, to increase the susceptibility of cellulose to hydrolysis. In some embodiments, the substrate is slurried prior to pretreatment.

The following references described various means of pretreatment. Steam explosion performing acid pretreatment of biomass substrates is described in U.S. Pat. No. 4,461,648. Continuous pretreatment using a slurry is described U.S. Pat. No. 7,754,457. Methods of alkali pretreatment is such as Ammonia Freeze Explosion, Ammonia Fiber Explosion or Ammonia Fiber Expansion (“AFEX”) are described in U.S. Pat. Nos. 5,171,592; 5,037,663; 4,600,590; 6,106,888; 4,356,196; 5,939,544; 6,176,176; 5,037,663 and 5,171,592. Alternative methods to AFEX utilizing a dilute ammonia pretreatments are described in WO2009/045651 and US 2007/0031953. Chemical pretreatments with organic solvents are disclosed in U.S. Pat. No. 4,556,430. Other pretreatments methods are disclosed in U.S. Pat. No. 7,465,791, and Weil et al. (1997) Appl. Biochem. Biotechnol., 68(1-2): 21-40 (1997).

Production Levels

The methods described herein produce fatty alcohols in high yield. Cells expressing FAR variants described herein may yield fatty alcohols in the range of about 0.5 g to at least 35.0 g fatty alcohols per liter of nutrient medium, depending upon the FAR variant polypeptide used. Exemplary culture conditions for E. coli are provided in the examples. Other E. coli culture conditions, as well as culture conditions for other host cells, are known or can be determined. In some embodiments, about 20 g/L to about 50 g/L (e.g., about 20 g/L, about 25 g/L, about 30 g/L, about 35 g/L, about 40 g/L, about 45 g/L, or about 50 g/L), or sometimes about 50 g/L to about 100 g/L (e.g., about 50 g/L, about 60 g/L, about 70 g/L, about 80 g/L, about 90 g/L, or about 100 g/L) are produced. In particular embodiments, the amount of fatty alcohols produced by the methods described herein is at least about 0.5 g/L, such as at least about 1 g/L, such as at least about 1.5 g/L, such as at least about 2.0 g/L, such as at least about 2.5 g/L, such as at least about 3 g/L, such as at least about 3.5 g/L, such as at least about 4 g/L, such as at least about 4.5 g/L, such as at least about 5 g/L, such as at least about 10 g/L of medium. In various embodiments, the amount of fatty alcohols produced by the methods described herein is at least about 20 g/L, such as at least about 30 g/L, such as at least about 40 g/L, such as at least about 50 g/L of medium. In some embodiments fermentation yields at least 0.1, at least 0.15 or at least 0.18 g fatty alcohol/gram glucose. In some embodiments fermentation yields at least 1 gram, at least 1.5 grams, or at least 1.8 grams fatty alcohol/gram dry cell weight.

In some embodiments, the methods described herein produce fatty alcohol compositions of particular chain lengths in high yield. In some embodiments, the methods described herein produce fatty alcohol compositions comprising at least about 90% C10-C18 fatty alcohols in an amount that is at least about 0.5 g/L, such as at least about 1 g/L, at least about 1.5 g/L, at least about 2.0 g/L, at least about 2.5 g/L, at least about 3 g/L, at least about 3.5 g/L, at least about 4 g/L, at least about 4.5 g/L, at least about 5 g/L, at least about 10 g/L, at least about 20 g/L, at least about 30 g/L, at least about 40 g/L, or at least about 50 g/L fatty alcohols per liter of medium. In some embodiments, the methods described herein produce fatty alcohol compositions comprising at least about 90% C12-C16 fatty alcohols in an amount that is at least about 0.5 g/L, such as at least about 1 g/L, at least about 1.5 g/L, at least about 2.0 g/L, at least about 2.5 g/L, at least about 3 g/L, at least about 3.5 g/L, at least about 4 g/L, at least about 4.5 g/L, at least about 5 g/L, at least about 10 g/L, at least about 20 g/L, at least about 30 g/L, at least about 40 g/L, or at least about 50 g/L fatty alcohols per liter of medium. In some embodiments, the methods described herein produce fatty alcohol compositions comprising at least about 90% C12-C14 fatty alcohols in an amount that is at least about 0.5 g/L, such as at least about 1 g/L, at least about 1.5 g/L, at least about 2.0 g/L, at least about 2.5 g/L, at least about 3 g/L, at least about 3.5 g/L, at least about 4 g/L, at least about 4.5 g/L, at least about 5 g/L, at least about 10 g/L, at least about 20 g/L, at least about 30 g/L, at least about 40 g/L, or at least about 50 g/L fatty alcohols per liter of medium. In some embodiments, the methods described herein produce fatty alcohol compositions comprising at least about 90% C14-C16 fatty alcohols in an amount that is at least about 0.5 g/L, such as at least about 1 g/L, at least about 1.5 g/L, at least about 2.0 g/L, at least about 2.5 g/L, at least about 3 g/L, at least about 3.5 g/L, at least about 4 g/L, at least about 4.5 g/L, at least about 5 g/L, at least about 10 g/L, at least about 20 g/L, at least about 30 g/L, at least about 40 g/L, or at least about 50 g/L fatty alcohols per liter of medium.

In some embodiments, the methods described herein produce an aggregate of the fatty alcohols C12:0 (1-dodecanol), C12:1 (cis Δ⁵-1-dodecenol), C14:0 (1-tetradecanol), C14:1 (cis Δ⁷-1-tetradecenol), C16:1 (cis Δ⁹-1-hexadecenol), C16:0 (1-hexadecanol), C18:1 (cis Δ¹¹-1-octadecenol), and C18:0 (1-octadecanol) in high yield. In some embodiments, the methods described herein produce an aggregate of the fatty alcohols C12:0 (1-dodecanol), C12:1 (cis Δ⁵-1-dodecenol), C14:0 (1-tetradecanol), C14:1 (cis Δ⁷-1-tetradecenol), C16:0 (1-hexadecanol), and C16:1 (cis Δ⁹-1-hexadecenol) in high yield. In some embodiments, the amount of such an aggregate of fatty alcohols that is produced is at least about 0.5 g/L, such as at least about 1 g/L, at least about 1.5 g/L, at least about 2.0 g/L, at least about 2.5 g/L, at least about 3 g/L, at least about 3.5 g/L, at least about 4 g/L, at least about 4.5 g/L, at least about 5 g/L, at least about 10 g/L, at least about 20 g/L, at least about 30 g/L, at least about 40 g/L, or at least about 50 g/L of medium.

In some embodiments, the amount of fatty alcohols produced by the methods described herein is in the range of about 100 mg/g to about 5 g/g of dry cell weight. In other embodiments, the amount of fatty alcohols produced by the methods described herein is in the range of about 1 g/g to about 4 g/g of dry cell weight, such as in the range of about 2 g/g to about 3 g/g of dry cell weight by routine modification of culturing conditions.

In certain embodiments, the amount of fatty alcohols produced by the methods described herein is in the range of about 10% to about 20% of dry cell weight, such as in the range of about 20% to about 30% of dry cell weight, such as in the range of about 30% to about 40% of dry cell weight, such as in the range of about 40% to about 50% of dry cell weight, such as in the in range of about 50% to about 60% of dry cell weight, such as in the range of about 60% to about 70% of dry cell weight, such as in the range of about 70% to about 80% of dry cell weight by routine modification of culturing conditions.

Recovery of Fatty Alcohols

Fatty alcohols produced by the methods can be isolated to yield fatty alcohol compositions. In some embodiments, recombinant microorganism hosts secrete the fatty alcohols into the nutrient medium. For cell-based methods carried out with recombinant microorganism hosts that secrete the fatty alcohols into the nutrient medium, the fatty alcohols can be isolated by solvent extraction of the aqueous nutrient medium with a suitable water immiscible solvent. Phase separation followed by solvent removal provides the fatty alcohol which may then be further purified and fractionated using methods and equipment known in the art. In other aspects of the disclosure, the secreted fatty alcohols coalesce to form a water immiscible phase that can be directly separated from the aqueous nutrient medium either during the fermentation or after its completion.

In certain embodiments, fatty alcohols are isolated by separating the cells from the aqueous nutrient medium, for example by centrifugation, resuspension and extraction of the fatty alcohols from the recombinant host cells using an organic solvent or solvent mixture. Suitable protocols for recovering fatty alcohols from recombinant host cells and/or culture medium are known to the skilled artisan.

In some embodiments, at least 10%, such as at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the fatty alcohols (e.g., C12 to 16 fatty alcohols or C12 to C14 fatty alcohols) produced by the methods described herein are secreted by the recombinant host cell or engineered microorganism comprising a FAR variant as described herein. In some embodiments, at least 10%, such as at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the fatty alcohols (e.g., C12 to 16 fatty alcohols or C12 to C14 fatty alcohols) produced by the methods described herein are secreted into the culture medium.

Fatty alcohols produced with microorganism hosts that do not secrete the fatty alcohols into the nutrient medium can be recovered by first lysing the cells to release the fatty alcohols and extracting the fatty alcohols from the lysate using conventional means. Reference is made to Yeast Protocols Handbook, Clontech Laboratories, Inc., A Takara Bio Company, 1290 Terra Bella Ave., Mountain View, Calif. 94043, published July 2009, available online.

VIII. Fatty Alcohol Derivatives

Fatty alcohols produced using the methods and variants disclosed herein can be converted to a variety of commercially useful compounds, referred to as fatty alcohol derivatives. Without limitation, exemplary fatty alcohol derivatives include fatty acids, fatty aldehydes, fatty esters, wax esters, fatty acetates, ethoxylates, sulphates, phosphates, amines, alkanes, and alkenes. The fatty alcohol derivatives may be obtained from fatty alcohols using either enzymatic or chemical methods. In some embodiments, the fatty alcohols can be reacted with a sulfonic acid group to produce sulfate derivatives.

In some embodiments, total fatty alcohols produced in a fermentation are derivatized. Sometimes fatty alcohols produced in a fermentation are fractionated, and a fraction(s) is derivatized.

Alkane and/or Alkene Compositions

In some embodiments, the fatty alcohol compositions produced by the methods described herein can be reduced to yield alkanes and/or alkenes having the same carbon chain length as the fatty alcohol starting materials. Without being bound by any particular theory, the hydroxyl group of an alcohol is a poor leaving group, and therefore, in principle a chemical moiety that binds to the oxygen atom of the hydroxyl group to make it a better leaving group can be used to reduce the fatty alcohols described herein. In another embodiment, alkanes can be produced by hydrogenation of fatty alcohols or fatty acids.

Any method known in the art can be used to reduce the fatty alcohols produced according to the methods described herein. In some embodiments, reduction of fatty alcohols can be carried out chemically, for example, by a Barton deoxygenation (or Barton-McCombie deoxygenation), a two-step reaction in which the alcohol is first converted to a methyl xanthate or thioimidazoyl carbamate, and the xanthate or thioimidazoyl carbamate is reduced with a tin hydride or trialkylsilane reagent under radical conditions to produce the alkane and/or alkene. See J. J. Li, C. Limberakis, D. A. Pflum, Modern Organic Synthesis in the Laboratory (Oxford University Press, 2007) at pp. 81-83.

In some embodiments, reduction of fatty alcohols to the corresponding alkanes and/or alkenes can be accomplished using a microorganism that has a biosynthetic pathway for reducing fatty alcohols. In certain embodiments, the microorganism is a bacterium. In specific embodiments, the bacterium is Vibrio furnissii strain M1. In some embodiments, the fatty alcohol compositions produced by the methods described herein are contacted with the appropriate microorganism for reduction to alkanes and/or alkenes. In other embodiments, the fatty alcohol compositions produced by the methods described herein are contacted with membrane fractions from the appropriate microorganism so that the reduction is carried out in a cell free system. See, e.g., Park, 2005, J. Bacteriol. 187(4):1426-1429.

In certain embodiments, alkanes and/or alkenes produced by the reduction of fatty alcohols described herein are isolated from the reaction mixture and unreduced fatty alcohol starting materials to produce a composition that comprises substantially all alkanes and/or alkenes. In some embodiments, the alkanes and/or alkenes produced by the reduction of fatty alcohols described herein and the unreacted fatty alcohol starting materials are isolated from the reaction mixture to produce a composition comprising alkanes and/or alkenes and fatty alcohols.

In certain embodiments, the resulting compositions comprise at least about 60% alkanes and/or alkenes, such as at least about 70% alkanes and/or alkenes, such as at least about 80% alkanes and/or alkenes, such as at least about 85% alkanes and/or alkenes, such as at least about 90% alkanes and/or alkenes, such as at least about 92% alkanes and/or alkenes, such as at least about 95% alkanes and/or alkenes, such as at least about 96% alkanes and/or alkenes, such as at least about 97% alkanes and/or alkenes, such as at least about 98% alkanes and/or alkenes, such as at least about 99% alkanes and/or alkenes by weight of the composition after reduction.

In other embodiments, the resulting compositions comprise at least about 10% alkanes and/or alkenes, such as at least about 20% alkanes and/or alkenes, such as at least about 30% alkanes and/or alkenes, such as at least about 40% alkanes and/or alkenes, such as at least about 50% alkanes and/or alkenes by weight of the composition after reduction.

In some typical embodiments, the compositions produced by the methods described herein comprise one or more alkanes selected from the group consisting of octane, decane, dodecane, tetradecane, hexadecane, octadecane, icosane and docosane. In other typical embodiments, the compositions produced by the methods described herein comprise one or more alkenes selected from the group consisting of octane, decene, dodecene, tetradecene, hexadecene, octadecene, icosene, and docosene.

In typical embodiments, C12 to C16 alkanes and/or alkenes comprise at least about 80%, such as at least about 85%, such as at least about 90%, such as at least about 92%, such as at least about 95%, such as at least about 97%, such as at least about 99% by weight of the total alkanes and/or alkenes in the composition. In certain embodiments, C12 to C14 alkanes and/or alkenes comprise about 80%, such as at least about 85%, such as at least about 90%, such as at least about 92%, such as at least about 95%, such as at least about 97%, such as at least about 99% by weight of the total alkanes and/or alkenes in the composition.

In certain embodiments, alkanes and/or alkenes having particular carbon chain lengths can be isolated from longer and/or shorter alkanes and/or alkenes, for example by HPLC. In certain embodiments, alkane and/or alkene compositions that are suitable, e.g., for use in jet fuels, comprise C10 to C14 alkanes and/or alkenes. In other embodiments, alkane and/or alkene compositions that are suitable, e.g., for use in diesel fuels comprise alkanes and/or alkenes that have 16 or more carbons (e.g., C16 or longer-chain alkanes and/or alkenes).

Ester Compositions

In certain embodiments, the fatty alcohols are further processed with a carboxylic acid to form acid esters. Esterification reactions of fatty alcohols are well-known in the art. In certain embodiments, the transesterification reaction is carried out in the presence of a strong catalyst, e.g., a strong alkaline such as sodium hydroxide. In other embodiments, the reaction is carried out enzymatically using an enzyme that catalyzes the conversion of fatty alcohols to acid esters, such as lipoprotein lipase. See, e.g., Tsujita et al., 1999, J. Biochem. 126(6):1074-1079.

IX. Exemplary Compositions Containing Fatty Alcohols and Fatty Alcohol Derivatives Detergent Compositions

In certain embodiments, the fatty alcohol compositions described herein and compounds derived there from can be used as components of detergent compositions. Detergent compositions containing fatty alcohols produced by the methods of the present invention include compositions used in cleaning applications, including, but not limited to, laundry detergents, hand-washing agents, dishwashing detergents, rinse-aid detergents, household detergents, and household cleaners, in liquid, gel, granular, powder, or tablet form. In some embodiments, the fatty alcohol compositions produced by the methods described above can be used directly in detergent compositions. In some embodiments, the fatty alcohols can be reacted with a sulfonic acid group to produce sulfate derivatives that can be used as components of detergent compositions. Detergent compositions that can be generated using the fatty alcohol compositions produced by the methods of the present invention include, but are not limited to, hair shampoos and conditioners, carpet shampoos, light-duty household cleaners, light-duty household detergents, heavy-duty household cleaners, and heavy-duty household detergents. Detergent compositions generally include, in addition to fatty alcohols, one or more or of builders (e.g., sodium carbonate, complexation agents, soap, and zeolites), enzymes (e.g., a protease, a lipase and an amylases); carboxymethyl cellulose, optical brighteners, fabric softeners, colourants and perfumes (e.g., cyclohexyl salicylate).

In some embodiments, sulfate derivatives derived from the fatty alcohol compositions are used in products such as hair shampoos, carpet shampoos, light-duty household cleaners, and light-duty household detergents. In some embodiments, fatty alcohol compositions (e.g., C16-C18) produced by the methods described herein are used in products such as hair shampoos and conditioners. In some embodiments, sulfate derivatives (e.g., C16-18) derived from the fatty alcohol compositions are used in products such as heavy-duty household cleaners and heavy-duty household detergents. Indeed, it is not intended that the present invention be limited to any particular detergent, detergent formulation nor detergent use.

Personal Care Compositions

In certain embodiments, the fatty alcohol compositions described herein and compounds derived therefrom are used as components of personal care compositions. In some embodiments, the fatty alcohol compositions produced by the methods described above can be used directly in personal care compositions. Personal care compositions containing fatty alcohols produced by the methods of the present invention include compositions used for application to the body (e.g., for application to the skin, hair, nails, or oral cavity) for the purposes of grooming, cleaning, beautifying, or caring for the body, including but not limited to lotions, balms, creams, gels, serums, cleansers, toners, masks, sunscreens, soaps, shampoos, conditioners, body washes, styling aids, and cosmetic compositions (e.g., makeup in liquid, cream, solid, anhydrous, or pencil form). In some embodiments, the fatty alcohols can be reacted with a sulfonic acid group to produce sulfate derivatives that can be used as components of said compositions. Indeed, it is not intended that the present invention be limited to any particular formulation, nor use.

In some embodiments, fatty alcohol compositions (e.g., C12) produced by the methods described herein are used in products such as lubricating oils, pharmaceuticals, and as an emollient in cosmetics. In some embodiments, fatty alcohol compositions (e.g., C14) produced by the methods described herein are used in products such as cosmetics (e.g., cold creams) for its emollient properties. In some embodiments, fatty alcohol compositions (e.g., C16) produced by the methods described herein are used in products such as cosmetics (e.g., skin creams and lotions) as an emollient, emulsifier, or thickening agent. In some embodiments, fatty alcohol compositions (e.g., C18) produced by the methods described herein are used in products such as lubricants, resins, perfumes, and cosmetics, e.g., as an emollient, emulsifier, or thickening agent. In some embodiments, sulfate derivatives (e.g., C12 to 14) derived from the fatty alcohol compositions produced by the methods described herein are used in products such as toothpastes. Indeed, it is not intended that the present invention be limited to any particular formulation, nor use.

Other Compositions

In some embodiments, fatty alcohol compositions (e.g., C12) produced by the methods described herein are used in products such as lubricating oils, pharmaceuticals, and as an emollient in cosmetics. In some embodiments, fatty alcohol compositions (e.g., C14) produced by the methods described herein are used in products such as cosmetics (e.g., cold creams) for its emollient properties. In some embodiments, fatty alcohol compositions (e.g., C16) produced by the methods described herein are used in products such as cosmetics (e.g., skin creams and lotions) as an emollient, emulsifier, or thickening agent. In some embodiments, fatty alcohol compositions (e.g., C18) produced by the methods described herein are used in products such as lubricants, resins, perfumes, and cosmetics, e.g., as an emollient, emulsifier, or thickening agent. In some embodiments, sulfate derivatives (e.g., C12 to 14) derived from the fatty alcohol compositions produced by the methods described herein are used in products such as toothpastes.

In some instances, fatty alcohols (especially cetyl alcohol, stearyl alcohol and myristyl alcohol) may be used as food additives (e.g., adjuvants and production aids).

X. Production and Recovery of Far Variants

In some cases, it will be useful to isolate a FAR variant polypeptide as described herein. Thus, in another aspect, the present invention provides a method of making a polypeptide having improved FAR enzymatic activity, for example, a polypeptide capable of catalyzing increased production of C12 and C14 fatty alcohols as compared to wild-type FAR. In some embodiments, the method comprises: providing a host cell transformed with any one of the described FAR polynucleotides of the present invention (e.g., a polynucleotide encoding a FAR variant polypeptide as described herein); culturing the transformed host cell in a culture medium under conditions in which the host cell expresses the encoded FAR variant polypeptide; and optionally recovering or isolating the expressed FAR variant polypeptide. The method further provides optionally lysing the transformed host cells after expressing the encoded FAR variant polypeptide and optionally recovering or isolating the expressed FAR variant polypeptide from the cell lysate. The present invention further provides a method of making an FAR variant polypeptide, said method comprising cultivating a host cell transformed with a FAR variant polypeptide under conditions suitable for the production of the FAR variant polypeptide and recovering the FAR variant polypeptide.

The FAR variant polypeptide can be recovered from the host cell using protein recovery techniques that are well known in the art, including those described herein. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract may be retained for further purification. Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, or other methods, which are well known to those skilled in the art.

The resulting polypeptide may be recovered/isolated and optionally purified by any of a number of methods known in the art. For example, the polypeptide may be isolated from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, chromatography (e.g., ion exchange, affinity, hydrophobic interaction, chromatofocusing, and size exclusion), or precipitation. Protein refolding steps can be used, as desired, in completing the configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed in the final purification steps. See, for example, Parry et al., 2001, Biochem. J. 353:117, and Hong et al., 2007, Appl. Microbiol. Biotechnol. 73:1331, both incorporated herein by reference. In addition to the references noted supra, a variety of purification methods are well known in the art, including, for example, those set forth in Sandana (1997) Bioseparation of Proteins, Academic Press, Inc.; Bollag et al. (1996) Protein Methods, 2^(nd) Edition, Wiley-Liss, NY; Walker (1996) The Protein Protocols Handbook Humana Press, NJ; Harris and Angal (1990) Protein Purification Applications: A Practical Approach, IRL Press at Oxford, Oxford, England; Harris and Angal Protein Purification Methods: A Practical Approach, IRL Press at Oxford, Oxford, England; Scopes (1993) Protein Purification: Principles and Practice 3^(rd) Edition, Springer Verlag, NY; Janson and Ryden (1998) Protein Purification: Principles, High Resolution Methods and Applications, Second Edition, Wiley-VCH, NY; and Walker (1998) Protein Protocols on CD-ROM, Humana Press, NJ, all of which are incorporated herein by reference.

XI. Examples

The following examples are offered to illustrate, but not to limit the claimed invention.

Example 1 Wild-type M. algicola DG893 FAR Gene Acquisition and Vector Construction

Gene acquisition of wild-type M. algicola DG893 FAR (“FAR Maa”) is described in the published application WO/2011/008535. The genomic sequence of M. algicola DG893 can also be found at GenBank Accession No. NZ_ABCP01000001.1. The amino acid sequence of the encoded FAR polypeptide is designated SEQ ID NO:2. A codon optimized polynucleotide sequence encoding the FAR polypeptide of SEQ ID NO: 2 is designated SEQ ID NO:1. The M. algicola DG893 FAR gene and genes encoding variants of the M. algicola DG893 FAR were cloned into the vector pCK110900 (depicted as FIG. 3 in U.S. Patent Appln. Pub. 2006/0195947) under the control of a lac promoter, as described in WO 2011/008535. The resulting plasmids were introduced into E. coli BW25113, BW25113 ΔfadE, or BW25113 Ltor R (Baba et al., Molecular Systems Biology, 2006 doi:10,1038/msb4100050 Article No. 2006.0008), W3110-ΔfhuA and MG1655-7740 by routine transformation methods.

Example 2 Evaluation of FAR Variants Using Microtiter Plate Assays

FAR variants were screened under several slightly differing conditions. Variant Nos. 1-144 were grown in 96-well shallow plates containing 180 μL Luria Bertani (LB) or M9YE medium supplemented with 1% glucose and 30 μg/mL chloramphenicol (CAM), for approximately 16-18 hours (overnight) in a shaker-incubator at 30° C., 200 rpm. A 5% inoculum was used in 96-deep-well plates to initiate fresh 380 μL culture containing 2×YT broth medium supplemented with 30 μg/mL CAM and 0.4% glucose. Some variants from later rounds of screening were grown in 96-well shallow plates containing 180 μL M9YE medium supplemented with 1% glucose and 30 μg/mL chloramphenicol (CAM), for approximately 16-18 hours (overnight) in a shaker-incubator at 30° C. or 37° C., 200 rpm. A 5% inoculum was used in 96-deep-well plates to initiate fresh 380 μL culture containing M9YE broth medium supplemented with 30 μg/mL CAM and 5% glucose. Other variants from later rounds of screening were grown in 96-well shallow plates containing 180 μL M9YE medium supplemented with 1% glucose, 150 mM BisTris pH 7.0, and 100 μg/mL spectinomycin (SPEC), for approximately 16-18 hours (overnight) in a shaker-incubator at 30° C. or 37° C., 200 rpm. A 5% inoculum was used in 96-deep-well plates to initiate fresh 380 μL culture containing M9YE broth medium supplemented with 100 μg/mL SPEC, 5% glucose, and 150 mM BisTris pH 7.0. The culture was incubated for 2 hours at 30° C. or 37° C., 250 rpm to an OD₆₀₀ of 0.6-0.8, at which point expression of the heterologous FAR gene was induced with isopropyl-β-D-thiogalactoside (IPTG) (1 mM final concentration). Incubation was continued for about 24 hours under the same conditions.

Cell cultures were extracted with 1 mL of isopropanol:methyl t-butyl ether (MTBE) (4:6 ratio) for 2 hours. The extracts were centrifuged and the upper organic phase was transferred into polypropylene 96-well plates and analyzed by the following GC-FID method using DB-5MS column (length 30 m, I.D. 0.32 mm, film 0.25 um): start temp. 150° C., increase the temperature at a rate of 25° C./min to 246° C. and hold for 2.66 min. Total run time, 6.50 min. Under the above GC conditions the approximate retention times (min (±0.05 min)) of produced fatty alcohols and acids were as follows: 2.50, C12:0-OH; 2.79, C12:0-OOH; 3.19, C14:0-OH; 3.48, C14:0-OOH; 3.18, C14:1-OH; 3.24, C14:0-OH; 3.61, C15:0-OH; 3.91, C16:1-OH; 3.98, C16:0-OH; 4.15, C16:0-OOMe (internal standard); 4.21, C16:1-OOH; 4.28, C16:0-OOH; 4.83, C18:1-OH; 4.92, C18:0-OH; 5.31, C18:0-OOH and 5.51, C18:1-OOH. Identification of individual fatty alcohol was done by comparison to commercial standards (Sigma Chemical Company, 6050 Spruce St. Louis, Mo. 63103).

Table 1 provides the relative fold improvement (FIOP) in the proportion of C12 and C14 fatty alcohols in the total fatty alcohols produced (i.e., the improvement in the percentage of C12 and C14 fatty alcohols in the total fatty alcohol titer) for illustrative variants relative to wild-type M. algicola DG893 FAR (SEQ ID NO 2) or a reference FAR variant. For the data shown in Table 1, the total fatty alcohol titer was determined by first adding the titers of each fatty alcohol measured (C10:0-OH, C12:1-OH, C12:0-OH, C13:0-OH, C14:1-OH, C14:0-OH, C15:0-OH, C16:1-OH, C16:0-OH, C18:1-OH, and C18:0-OH). The percentage of each fatty alcohol species was then calculated as a percentage of the total fatty alcohols measured.

For the data shown in Table 1, codon-optimized SEQ ID NO:1 was mutated and used to express FAR variants. Relative improvement in the proportion of C12 and C14 fatty alcohol produced is presented as fold improvement (FIOP) over SEQ ID NO:2 (for Variant Nos. 1-26); over FAR Variant 26 (for Variant Nos. 27-85); over FAR Variant 85 (for Variant Nos. 86-92); over FAR Variant 92 (for Variant Nos. 93-118); or over FAR Variant 118 (for Variant Nos. 119-143) at 30° C. In Table 1, the amino acid substitutions listed for each variant correspond to residue positions of SEQ ID NO:2 (e.g., “N134S” means that the residue at position 134 in SEQ ID NO:2 (asparagine) is substituted with serine), and the amino acid positions were determined by optimal alignment with SEQ ID NO:2.

TABLE 1 Variant FAR polypeptides and relative fold improvement in proportion of C12 and C14 fatty alcohols produced FIOP in proportion Variant of C12 and C14 fatty No. Amino acid substitutions relative to SEQ ID NO: 2 alcohols produced^(a) 1 A2H ++ 2 E71Q + 3 T246A + 4 A2T + 5 A2Q + 6 Q7N + 7 E137L + 8 A2P + 9 A2W + 10 A2D + 11 G9F + 12 A511R + 13 S331V + 14 A2F + 15 L209T + 16 S74K + 17 A2G + 18 A511G + 19 A443T + 20 P188S + 21 N134R + 22 N134S + 23 N134K + 24 E227G + 25 E138Q + 26 N134S; E138Q; P188S; A511T ++ 27 N134S; E138Q; P188S; S306W; A511T *** 28 N134S; E138Q; P188S; E421R; A511T ** 29 N134S; E138Q; P188S; P405L; A511T ** 30 N134S; E138Q; P188S; P405C; A511T ** 31 N134S; E138Q; P188S; P405V; A511T ** 32 N134S; E138Q; P188S; P405A; A511T ** 33 N134S; E138Q; P188S; A412V; A511T ** 34 N134R; E138Q; P188S; A511T ** 35 N134K; E138Q; P188S; A511T ** 36 E138Q; P188S; A511T ** 37 N134S; E138Q; P188S; G410R; A511T ** 38 N134S; E138Q; P188S; L502S; A511T ** 39 N134S; E138Q; P188S; E421I; A511T ** 40 N134S; E138Q; P188S; P405F; A511T ** 41 N134S; E138Q; P188S; P405G: A511T ** 42 N134S; E138Q; P188S; G487Y; A511T * 43 N134S; E138Q; P188S; D429K; A511T * 44 N134S; E138Q; P188S; G401V; A511T * 45 N134S; E138Q; P188S; E421S; A511T * 46 N134S; E138Q; P188S; E421L; A511T * 47 N134S; E138Q; P188S; G401L; A511T * 48 N134S; E138Q; P188S; L499S; A511T * 49 N134S; E138Q; P188S; Q418R; A511T * 50 N134S; E138Q; P188S; E303G; A511T * 51 N134S; E138Q; P188S; Q418V; A511T * 52 N134S; E138Q; P188S; Q418I; A511T * 53 N134S; E138Q; P188S; E421N; A511T * 54 N134S; E138Q; P188S; E421V; A511T * 55 N134S; E138Q; P188S; A505K; A511T * 56 N134S; E138Q; P188S; L209N; A511T * 57 N134S; E138Q; P188S; G401S; A511T * 58 N134S; E138Q; P188S; L502R; A511T * 59 N134S; E138Q; P188S; R508G; A511T * 60 N134S; E138Q; P188S; S433H; A511T * 61 N134S; E138Q; P188S; A511S * 62 N134S; E138Q; P188S; A374Y; A511T * 63 N134S; E138Q; S161P; P188S; A511T * 64 N134S; E138Q; P188S; G401A; A511T * 65 N134S; E138Q; P188S; R508H; A511T * 66 N134S; E138Q; P188S; S433N; A511T * 67 N134S; E138Q; P188S; L502A; A511T * 68 N134S; E138Q; P188S; L502Q; A511T * 69 N134S; E138Q; P188S; A416L; A511T * 70 N134S; E138Q; P188S; S433K; A511T * 71 N134S; E138Q; L148E; P188S; A511T * 72 N134S; E138Q; P188S; Y380R; A511T * 73 E87V; N134S; E138Q; P188S; A511T * 74 N134S; E138Q; P188S; A409V; A511T * 75 N134S; E138Q; P188S; T430I; A511T * 76 V77I; N134S; E138Q; P188S; A511T * 77 N134S; E138Q; P188S; Y500N; A511T * 78 N134S; E138Q; Q180R; P188S; A511T * 79 N134S; E138Q; P188S; V404I; A511T * 80 N134S; E138Q; P188S; E288Q; A511T * 81 N134S; E138Q; P188S; K510D; A511T * 82 N134S; E138Q; P188S; S433Y; A511T * 83 N134S; E138Q; V185I; P188S; A511T * 84 N134S; E138Q; P188S; A416V; A511T * 85 N134S; E138Q; P188S; P405V; Q418V; A511T *** 86 N134S; E138Q; P188S; P405V; Q418R; S433R; A511T † 87 N134S; E138Q; P188S; P405V; Q418V; S433R; A511T † 88 H61R; N134S; E138Q; P188S; P405V; Q418V; A511T † 89 N134S; E138Q; P188S; P405V; Q418V; K509H; A511T † 90 N134S; E138Q; P188S; P405V; Q418V; R508D; A511T † 91 N134S; E138Q; P188S; P405V; Q418V; K509D; A511T † 92 N134S; E138Q; P188S; P405V; Q418V; S458Q; L502S; † R508D; K509D; A511T 93 N134S; E138Q; P188S; A389I; P405V; Q418V; S458Q; {circumflex over ( )}{circumflex over ( )}{circumflex over ( )} L502S; R508D; K509D; A511T 94 N134S; E138Q; P188S; A389M; P405V; Q418V; {circumflex over ( )}{circumflex over ( )} S458Q; L502S; R508D; K509D; A511T 95 N134S; E138Q; P188S; A389L; P405V; Q418V; S458Q; {circumflex over ( )}{circumflex over ( )} L502S; R508D; K509D; A511T 96 N134S; E138Q; P188S; A389V; P405V; Q418V; S458Q; {circumflex over ( )}{circumflex over ( )} L502S; R508D; K509D; A511T 97 V104I; N134S; E138Q; P188S; P405V; Q418V; S458Q; {circumflex over ( )}{circumflex over ( )} L502S; R508D; K509D; A511T 98 V104M; N134S; E138Q; P188S; P405V; Q418V; {circumflex over ( )} S458Q; L502S; R508D; K509D; A511T 99 N134S; E138Q; P188S; S283M; P405V; Q418V; {circumflex over ( )} S458Q; L502S; R508D; K509D; A511T 100 N134S; E138Q; P188S; S283F; P405V; Q418V; S458Q; {circumflex over ( )} L502S; R508D; K509D; A511T 101 N134S; E138Q; P188S; S283E; P405V; Q418V; S458Q; {circumflex over ( )} L502S; R508D; K509D; A511T 102 N134S; E138Q; P188S; S283T; P405V; Q418V; S458Q; {circumflex over ( )} L502S; R508D; K509D; A511T 103 N134S; E138Q; P188S; T370I; P405V; Q418V; S458Q; {circumflex over ( )} L502S; R508D; K509D; A511T 104 N134S; E138Q; P188S; P405V; Q418V; D429R; {circumflex over ( )} S458Q; L502S; R508D; K509D; A511T 105 N134S; E138Q; P188S; P405V; Q418V; D429E; {circumflex over ( )} S458Q; L502S; R508D; K509D; A511T 106 G14V; N134S; E138Q; P188S; P405V; Q418V; S458Q; {circumflex over ( )} L502S; R508D; K509D; A511T 107 N134S; E138Q; P188S; Q377K; P405V; Q418V; {circumflex over ( )} S458Q; L502S; R508D; K509D; A511T 108 N134S; E138Q; P188S; S244A; P405V; M413R; {circumflex over ( )} Q418V; S458Q; L502S; R508D; K509D; A511T 109 N134S; E138Q; P188S; S244P; P405V; Q418V; S458Q; {circumflex over ( )} L502S; R508D; K509D; A511T 110 N134S; E138Q; P188S; P405V; Q418V; R432C; {circumflex over ( )} S458Q; L502S; R508D; K509D; A511T 111 N134S; E138Q; P188S; P405V; Q418V; S458Q; L502S; {circumflex over ( )} R508D; K509D; A511T 112 L69E; N134S; E138Q; P188S; P405V; Q418V; S458Q; {circumflex over ( )} L502S; R508D; K509D; A511T 113 G14R; N134S; E138Q; P188S; P405V; Q418V; S458Q; {circumflex over ( )} L502S; R508D; K509D; A511T 114 N134S; E138Q; P188S; D376P; P405V; Q418V; {circumflex over ( )} S458Q; L502S; R508D; K509D; A511T 115 N134S; E138Q; P188S; P405V; Q418V; S458Q; {circumflex over ( )} Q474R; L502S; R508D; K509D; A511T 116 N134S; E138Q; P188S; P405V; Q418V; S458Q; {circumflex over ( )} V466Q; L502S; R508D; K509D; A511T 117 L69Q; N134S; E138Q; P188S; P405V; Q418V; S458Q; {circumflex over ( )} L502S; R508D; K509D; A511T 118 N134S; E138Q; P188S; P405V; Q418V; S433K; S458Q; {circumflex over ( )}{circumflex over ( )}{circumflex over ( )} L502S; R508D; K509D; A511T 119 N134S; E138Q; P188S; P405V; M365N; Q418V; # S433K; S458Q; L502S; R508D; K509D; A511T 120 N134S; E138Q; P188S; P405V; Q418I; S433K; S458Q; # L502S; R508D; K509D; A511T 121 H98P; N134S; E138Q; P188S; P405V; Q418V; S433K; # S458Q; L502S; R508D; K509D; A511T 122 N134S; E138Q; P188S; P405V; Q418V; R432Q; # S433K; S458Q; L502S; R508D; K509D; A511T 123 T91R; N134S; E138Q; P188S; P405V; Q418V; S433K; # S458Q; L502S; R508D; K509D; A511T 124 N134S; E138Q; V1531; P188S; P405V; Q418V; S433K; # S458Q; L502S; R508D; K509D; A511T 125 N134S; E138Q; P188S; P405V; Q418V; S433K; S458Q; ## G487R; L502S; R508D; K509D; A511T 126 N134S; E138Q; P188S; K224R; P405V; Q418V; S433K; # S458Q; L502S; R508D; K509D; A511T 127 N134S; E138Q; P188S; P405V; Q418V; T430H; S433K; # S458Q; L502S; R508D; K509D; A511T 128 N134S; E138Q; P188S; V398Y; P405V; Q418V; S433K; # S458Q; L502S; R508D; K509D; A511T 129 Q18I; R65G; N128H; N134S; E138Q; N177T; P188S; ## K224R; L226M; P405V; Q418V; S433K; S458Q; G487R; L502S; R508D; K509D; A511T 130 N134S; E138Q; P188S; G401C; P405V; Q418V; # S433K; S458Q; L502S; R508D; K509D; A511T 131 N134S; E138Q; P188S; P405V; Q418V; S433K; S452N; # S458Q; L502S; R508D; K509D; A511T 132 N134S; E138Q; P188S; P405V; Q418V; E421P; S433K; # S458Q; L502S; R508D; K509D; A511T 133 N134S; E138Q; P188S; P405V; L406Y; Q418V; S433K; ## S458Q; L502S; R508D; K509D; A511T 134 N134S; E138Q; P188S; G351C; P405V; Q418V; # S433K; S458Q; L502S; R508D; K509D; A511T 135 N128H; N134S; E138Q; P188S; P405V; Q418V; S433K; ## S458Q; L502S; R508D; K509D; A511T 136 N134S; E138Q; P188S; P405V; A409Y; Q418V; S433K; # S458Q; L502S; R508D; K509D; A511T 137 R65G; N134S; E138Q; P188S; P405V; Q418V; S433K; # S458Q; L502S; R508D; K509D; A511T 138 N134S; E138Q; P188S; P405V; Q418V; S433K; S458Q; ## L502S; R508D; K509D; A511K 139 N134S; E138Q; P188S; V207L; P405V; Q418V; S433K; # S458Q; L502S; R508D; K509D; A511T 140 N134S; E138Q; P188S; P405V; A409W; Q418V; # S433K; S458Q; L502S; R508D; K509D; A511T 141 N134S; E138Q; P188I; P405V; Q418V; S433K; S458Q; # L502S; R508D; K509D; A511T 142 N134S; E138Q; P188S; P405V; Q418V; S433K; S452G; # S458Q; L502S; R508D; K509D; A511T 143 N134S; E138Q; P188S; P405V; G410H; Q418V; # S433K; S458Q; L502S; R508D; K509D; A511T ^(a)Fatty alcohols measured for the relative fold improvement include: C12:1 (cis Δ⁵-1-dodecenol), C12:0 (1-dodecanol), C14:1 (cis Δ⁷-1-tetradecanol), and C14:0 (1-tetradecanol). + = 1.0 to 1.5 fold improvement over wild-type M. algicola FAR ++ = 1.6 to 2.0 fold improvement over wild-type M. algicola FAR * = 1.0 to 1.5 fold improvement over Variant No. 26 ** = 1.6 to 2.0 fold improvement over Variant No. 26 *** = >2.0 fold improvement over Variant No. 26 † = 1.0 to 1.5 fold improvement over Variant No. 85 {circumflex over ( )} = 0.5 to 1.0 fold improvement over Variant No. 92 {circumflex over ( )}{circumflex over ( )} = >1.0 to 2.0 fold improvement over Variant No. 92 {circumflex over ( )}{circumflex over ( )}{circumflex over ( )} = >2.0 fold improvement over Variant No. 92 # = 0.5 to 1.0 fold improvement over Variant No. 118 ## = >1.0 fold improvement over Variant No. 118

Table 2 provides the amount by percentage of C12 and C14 fatty alcohols in the total fatty alcohol titer (as measured in g/L) and the relative fold improvement (FIOP) in the proportion of C12 and C14 fatty alcohols in the total fatty alcohols produced (i.e., the improvement in the percentage of C12 and C14 fatty alcohols in the total fatty alcohol titer) for illustrative variants relative to Variant No. 129 (SEQ ID NO:10) at 30° C. Fatty alcohols were measured, and the proportion of each fatty alcohol species was determined, as described above for Table 1. In Table 2, the amino acid substitutions listed for each variant correspond to residue positions of SEQ ID NO:10. Variants were screened as described above. For the variants illustrated in Table 2, the titer (in g/L) of C12 and C14 fatty alcohols produced was measured to be in the range of 0.58 to 1.72 g/L. The relative fold improvement in the proportion of C12 and C14 fatty alcohols produced, relative to SEQ ID NO:10, ranged from 0.4-fold to 1.2-fold.

TABLE 2 Variant FAR polypeptides and the % of C12 and C14 fatty alcohol production and relative improvement in proportion of C12 and C14 fatty alcohols produced FIOP in proportion Variant % C12 of C12 and C14 fatty No. Amino Acid Substitutions Relative to SEQ ID NO: 10 and C14 alcohols produced 144 G65R, S266A, R382H, A389M, G401V 41 ++ 145 G65R, S266A, A389M, A412V 41 ++ 146 G65R, S266A, A389M, A412V, T511K 39 ++ 147 G65R, S266A, A389M, G401V, A412V, T511K 40 ++ 148 G65R, S266A, A389M, G401V, T511K 40 ++ 149 G65R, S266A, A389M, G401V, A412V 40 ++ 150 I18Q, S74P, S266A, G410R, A505K 38 ++ 151 S306W, V405C, E421G, R487Y 40 ++ 152 I18Q, S74P, S266A, T370I, G410R 38 ++ 153 I18Q, S74P, S266A, T370I, G410R, A505K 36 ++ 154 S266A, G401V 36 ++ 155 V104I, V405C 36 ++ 156 I18Q, S266A, T370I, G410R 35 + 157 S74P, S266A, T370I 37 ++ 158 G65R, S266A, G401V, T511K 32 + 159 I18Q, S74P, S266A, T370I 34 + 160 S74P, S266A, T370I, G410R, A505K 38 ++ 161 I18Q, V104I, S134R, S306W, V405C, E421G 36 ++ 162 I18Q, S74P, S266A, G410R, E421S, A505K 36 ++ 163 I18Q, V104I, S134R, S306W, V405C, E421G, R487Y 36 ++ 164 S266A, A389M, A412V, T511K 38 ++ 165 I18Q, V104I, S306W, V405L, E421R, R487Y 38 ++ 166 I18Q, V104I, S134K, S306W, V405C, R487Y 35 + 167 A412V, D429K, L499R 33 + 168 I18Q, S74P, S266A, T370I, A505K 32 + 169 S74P, S266A, A505K 35 ++ 170 S134K, S306W, V405L 35 + 171 2G65R, S266A 30 + 172 18Q, V104I, S134K, S306W, V405L 34 + 173 I18L, S74P, S266A, T370I, E421R, A505K 36 ++ 174 V77I, T246A, A374Y, V405A, R487G, D508G 31 + 175 I18Q, V104I, S306W, V405L 34 + 176 S266A, A389M 38 ++ 177 V77I, T246A, V405A, R487G, D508G 31 + 178 I18Q, S74P, S266A, T370I, G410R, E421S, A505K 33 + 179 I18Q, V104I, S134R, S306W, V405C 33 + 180 H61R, V77I, T246V, V405C, D508G 34 + 181 T246A, A374Y, V405A, R487G, D508G 30 + 182 I18Q, V1041, S134R, S306W, V405C, E421R, R487Y 34 + 183 I18Q, S266A, T370I, G410R, E421S, A505K 35 + 184 I18Q, S74P, S266A, T370I, E421R, A505K 35 + 185 I18Q, S266A, T370I, G410R, A505K 32 + 186 I18Q, S134R, S306W, V405C 31 + 187 S161P, S283F, A412V, D429K, L499R 30 + 188 I18Q, S74P, S266A, T370I, E421S, A505K 32 + 189 E71Q, E137L, S161P, S283F, A412V, L499R 27 + 190 H61R, T246A, V405A, R487G, D508G 27 + 191 I18Q, S74P, S266A, T370I, R382H, G410R, S502W, A505K 26 + 192 I18Q, S306W, V398Y, V405C, E421R 42 ++ 193 E71Q, E137L, S161P, S283F, L499S 26 + 194 V104I, S134R, S306W, V405C, R487Y 30 + 195 E71Q, E137L, S161P, S283F, D429K, L499S 26 + 196 S266A, A389M, G401V, A412V, T511K 34 + 197 H61R, T246A, A374Y, V405C, R487G, D508G 24 + 198 I18Q, V104I, S134K, S306W, V398Y, V405C, E421R 39 ++ 199 E71Q, E137L, S161P, S283F, Y380R, A412V, D429R 25 + 200 E71Q, E137L, S161P, S283F, Y380R, A412V, Y446F, L499R 21 + * % value rounded to the nearest unit. + = ≦1.0 fold improvement over Variant No. 129 ++ = >1.0 fold improvement over Variant No. 129

Example 3 Chain Length Profile of Fatty Alcohols Exhibited by Representative FAR Variants

The chain length profile of a subset of FAR variants was evaluated. Table 3 provides the relative chain length distribution of fatty alcohols exhibited by recombinant E. coli strains expressing wild-type FAR Maa or FAR variants when cultured at 30° C. As described above for Example 2, the total fatty alcohol titer was determined by adding the titers of each fatty alcohol measured (C10:0-OH, C12:1-OH, C12:0-OH, C13:0-OH, C14:1-OH, C14:0-OH, C15:-OH, C16:1-OH, C16:0-OH, C18:1-OH, and C18:0-OH). The percentage of each fatty alcohol species was then calculated as a percentage of the total fatty alcohols measured.

TABLE 3 Relative chain length distribution of fatty alcohols FAR Variant Relative Chain Length Distribution of fatty alcohols^(a) No. C12:0 C14:0 C14:1 C16:1 C16:0 C18:1 Wild-type 0 3 0 10 36 51 (SEQ ID NO: 2) Variant 0 4 0 23 31 42 26 Variant 0 9 0 37 28 26 85 Variant 0 9 0 28 38 25 92 Variant 0 19 0 40 31 10 118 Variant 1 29 4 52 12 3 129 ^(a)The relative chain length distribution is expressed as a percentage of the total fatty alcohols (g/L) detected via GC-FID. Fatty alcohols include: C12:0 (1-dedecanol), C14:1 (cis Δ⁷-1-tetradecanol), C14:0 (1-tetradecanol), C16:1 (cis Δ⁹-1-hexadecenol), C16:0 (1-hexadecanol), and C18:1 (cis Δ¹¹-1-octadecenol).

Example 4 Evaluation of FAR Variants with Improved Activity Using Fermentors

In an aerated, agitated stirred tank 10 L fermentor, 3.0 L of growth medium containing 33.85 g 5×M9 powder (BD Difco), 6 g Bacto yeast extract (BD), 3 g ammonium phosphate dibasic (Sigma-Aldrich), 15 g ammonium sulfate (EMD), 9 g glucose (Sigma), 1.48 g magnesium sulfate, heptahydrate (Sigma), 44 mg Calcium chloride, Dihydrate (Sigma), 15 ml trace elements solution, 12.6 mg EDTA (Sigma-Aldrich), 150 mg Fe(III) Citrate (Sigma), 6.75 mg Thiarnine.HCl (Sigma), and 90 μg chloroamphenicol (Sigma Chemical Co.) was brought to a temperature of 30° C. or 37° C. The fermentor is inoculated with a culture of E. coli strain containing FAR variants to a starting optical density (OD₆₀₀) of about 1.0. The inoculum was grown in a 1000 mL baffled shake flask containing 200 ml of 47.6 g/L terrific broth powder (Difco), 4 ml/L glycerol (Sigma), and 30 μg/ml chloroamphenicol (Sigma Chemical Co.) at 30° C. or 37° C., 250 rpm until the OD600 reached ˜8.0-10.0. The fermentor was agitated at 300-1200 rpm and air supplied at 3.0 L/min to maintain a minimum dissolved oxygen level of 30% of saturation. The pH of the culture was controlled at about 7.0 by addition of 5 N sodium hydroxide.

After consumption of the 3 g/L initial glucose, an exponential fed-batch growth phase was initiated by exponential addition of feed solution containing 500 g/L glucose (Sigma), 13.06 g/L magnesium sulfate, heptahydrate (Sigma), 100 g/L ammonium sulfate (EMD), 10 ml/L trace elements solution, 8.4 mg/L EDTA (Sigma-Aldrich), 100 mg/L Fe(III) Citrate (Sigma), 4.5 mg/L Thiamine.HCl (Sigma), and 30 μg/L chloroamphenicol (Sigma Chemical Co.) to the fermentor. After approximately 16 hours of fed-batch culture, the expression of FAR variants was induced by the addition of isopropyl-β-D-thiogalactoside (IPTG) (US Biological) to a final concentration of about 1 mM. Production of fatty alcohol was maintained by addition of a feed solution containing 650 g/L glucose (Sigma), 5.6 g/L magnesium sulphate, heptahydrate (Sigma), 6.5 g/L ammonium sulfate (EMD), 15 ml/L trace elements solution, 6.3 mg/L EDTA (Sigma-Aldrich), 75 mg/L Fe(III) Citrate (Sigma), 5.6 mg/L Thiamine.HCl (Sigma), 30 μg/L chloroamphenicol (Sigma Chemical Co.), and 1 mM IPTG. The culture was grown for about another 120 hours at 30° C. or 37° C. Samples were taken at various time points for extraction and analysis. Extraction and quantification of fatty alcohols was performed as described above in Example 2.

Example 5 Generation of FAR Variants SEQ ID NO:16 and SEQ ID NO:18 and Fatty Alcohol Production with SEQ ID NOs:16 and 18

Libraries of saturation mutagenesis of whole protein as well as combinatorial libraries were created in FAR from M. algicola and screened in E. coli for fatty alcohol production and chain length composition via GC-FID. Combinations of amino acid substitutions were identified that yielded an increased proportion of 1-dodecanol and 1-tetradecanol in the total fatty alcohol titer compared to backbone, including the following combinations of substitutions listed in Table 4:

TABLE 4 Variant FAR polypeptides and the relative fold improvement in proportion of C12 and C14 fatty alcohols produced FIOP in proportion of C12 and C14 FAR Amino acid substitutions relative fatty alcohols Sequence to SEQ ID NO: 2 produced^(a) SEQ ID Q7N; Q18I; R65G; N128H; E138Q; N177T; ++ NO: 16 P188S; K224R; L226M; E227G; M365N; G401V; P405V; Q418V; S433K; S458Q; G487R; L502S; R508D; K509D; A511T SEQ ID Q7N; Q18I; V104I; N128H; E138Q; N177T; ++ NO: 18 P188S; K224R; L226M; E227G; M365N; G401V; P405V; G410R; Q418V; S433K; S458Q; G487R; L502S; R508D; K509D; A511T ^(a)FIOP in % of C12 and C14 for SEQ ID NO: 16 was measured over Variant 129; FIOP in % of C12 and C14 for SEQ ID NO: 18 was measured over SEQ ID NO: 16. ++ = >1.0 fold improvement over backbone sequence

The FAR variant polynucleotide of SEQ ID NO:15, encoding the FAR variant polypeptide having the amino acid sequence of SEQ ID NO:16, was introduced into the vector pCDX11 to generate pCDX11-SEQ ID NO:16. The FAR variant polynucleotide of SEQ ID NO:17, encoding the FAR variant polypeptide having the amino acid sequence of SEQ ID NO:18, was introduced into the vector pCDX11 to generate pCDX11-SEQ ID NO:18. To obtain a tightly regulated expression vector, the P_(TRC) promoter present in pLS8379 was replaced with a synthetic DNA fragment containing a P_(TRC) variant where a symmetrical Lac operator (Sadler et al., 1983, PNAS 80:6785-6789) was introduced upstream of the −35 region of P_(TRC). This promoter was synthesized as an EcoRV-NcoI DNA fragment (GeneScript, Piscataway, N.J.) (SEQ ID NO:45) and used to replace the EcoRV-NcoI region from pLS8379 previously cut with the same restriction enzymes. The DNA sequence of plasmid pLS8379 is provided as SEQ ID NO:46. A ligation reaction containing the two DNA fragments was incubated overnight at 16° C. and then transformed into E. coli Top10 electrocompetent cells (Invitrogen, Carlsbad, Calif.) following the manufacturer's protocols. Cells were plated onto LB agar plates containing 100 micrograms/mL of spectinomycin. Places were then incubated overnight at 37° C. Obtained clones were sequence verified.

Recombinant E. coli host strains comprising a plasmid as specified in Table 5 or Table 6 below were grown in M9YE (Sambrook et al., (2001) Molecular Cloning: A Laboratory Manual 3^(rd) Ed Cold Spring Harbor, N.Y.) medium supplemented with 1% glucose, 2 g/l yeast extract and 100 μg/mL spectinomycin for approximately 16-18 hours (overnight) at 30° C., 200 rpm. A 5% inoculum was used to initiate fresh M9YE media, 5% glucose and 2 g/l yeast extract containing 30 30 μg/nL CAM. The culture was incubated in a shaker for 2.5 hours at 30° C. and at 250 rpm to an OD₆₀₀ of about 0.6 to about 0.8. The expression of the heterologous FAR was then induced with isopropyl-β-D-thiogalactoside (IPTG) (1 mM final concentration). Incubation was continued for about 48 hours under the same conditions. Fatty acid species including fatty alcohols were extracted using 1 mL of methyl isobutyl ketone (MIBK) into 500 μl of cell culture, sealed tightly and shaken for ≧2.5 hr. The extract was centrifuged and analyzed directly by GC-FID. A 1 μL sample was analyzed by GC-FID with the split ratio 1:10 using the following conditions: GC-6890N from Agilent Technologies equipped with FID detector and HP-5 column (length 30 m, I.D. 0.32 mm, film 0.25 μm). GC method: start at 100° C., increase the temperature with a rate of 25° C./min to 246° C. and hold for 1.96 min. Total run time was 7.8 min. Under the above GC conditions, the approximate retention times (min) of produced fatty alcohols and acids were as follows: 1.81, C10:0-OH; 2.47, C12:0-OH; 5.08, C14:0-OH; 5.40, C14:0-OOH; 5.74, C16:1-OH; 5.93, C16:0-OH; 6.11, C16:0-OOMe (internal standard); 6.16, C16:1-OOH; 6.29, C16:0-OOH; 6.80, C18:1-OH; 6.90, C18:0-OH; and 7.3, C18:0- and C18:1-OOH. Results of fatty alcohol production (total fatty alcohol production (“FOH”) and the relative percentages of C12, C14, C16, or C18 fatty alcohols) under these conditions are depicted in Tables 5-6. Identification of individual fatty alcohols was determined by comparison to commercial standards (Sigma Chemical Company, 6050 Spruce St. Louis, Mo. 63103).

TABLE 5 Fatty Alcohol (FOH) Production in a W3110 ΔfhuA Strain % % % % % Total FOH Plasmids Saturation C12 C14 C16 C18 (g/L)* pCDX11-SEQ ID 65 12 57 30 2 +++ NO: 16 % as measured by calculating the individual fatty alcohols as part of the sum of all fatty alcohol measured (C10:0-OH, C12:0-OH, C14:0-OH, C16:1-OH, C16:0-OH, C18:1-OH, and C18:0-OH). All values were rounded to the closest unit. CX (wherein X = 12, 14, 16 or 18) denotes both saturated and unsaturated species. *+++ = ≧3.0.

TABLE 6 Fatty Alcohol (FOH) Production in a W3110K Strain % % % % % Total FOH Plasmids saturation C12 C14 C16 C18 (g/L) pCDX11-SEQ ID 56 13 52 30 2 ++ NO: 18 % as measured by calculating the individual fatty alcohols as part of the sum of all fatty alcohol measured (C10:0-OH, C12:0-OH, C14:0-OH, C16:1-OH, C16:0-OH, C18:1-OH, and C18:0-OH). All values were rounded to the closest unit. CX (wherein X = 12, 14, 16 or 18) denotes both saturated and unsaturated species. *++ = 1.0 to <3.0.

Example 6 Evaluation of FAR Variants

E. coli strains containing M. algicola FAR were grown in 96-well NUNC flat well plates containing 180 μL M9YE medium supplemented with 1% glucose, 150 mM BisTris pH 7.0, and 100 mg/L spectinomycin. The plates were incubated for 24 hours at 30° C., 200 rpm, 2″ throw, and 85% relative humidity. The cells were diluted by transferring 20 μL of overnight grown culture into the 96-well CoStar deep well plates containing 400 μL M9YE supplemented with a total of 5% glucose, 150 mM BisTris pH 7.0, and 100 mg/L spectinomycin. The strains were induced with 1 mM IPTG 6 hours post induction and were incubated for 48 hours at 30° C., 250 rpm, 2″throw, and 85% relative humidity. Cell cultures were extracted with 950 μL of methyl isobutyl ketone for 2 hours. The extracts were centrifuged and the upper organic phase was transferred into polypropylene 96-well plates and analyzed using GC-FID. A 4 μL sample was analyzed by GC-FID with a split ratio of 1:10 using the following conditions: GC-6890N from Agilent Technologies equipped with a FID detector and HP-5 column (length 30 m, I.D. 0.32 mm, film 0.25 um): start temp. 150° C., increase the temperature at a rate of 25° C./min to 246° C. and hold for 2.66 min. Total run time, 6.50 min. Under the above GC conditions the approximate retention times (min (±0.05 min)) of produced fatty alcohols and acids were as follows: 2.41, C12:0-OH; 3.04, C14:1Δ7-OH, 3.14; C14:0-OH; 3.53, C15:0-OH; 3.86, C16:1A9-OH, 3.93; C16:0-OH; 4.75, C18:1A11-OH, 4.85; C18:0-OH. Identification of individual fatty alcohol was done by comparison to commercial standards (Sigma Chemical Company, 6050 Spruce St. Louis, Mo. 63103).

Table 7 provides the relative fold improvement (FIOP) in the proportion of C12:0 fatty alcohols produced and in the increase in total fatty alcohol titer for illustrative variants relative to FAR variant SEQ ID NO:18 at 30° and/or 37° C. In Table 7, the amino acid substitutions listed for each variant correspond to residue positions of SEQ ID NO:18.

TABLE 7 Variant FAR polypeptides and relative fold improvement in proportion of C12:0 fatty alcohols produced and total fatty alcohol titer Amino Acid FIOP in FIOP in FIOP in FIOP in Substitutions proportion total fatty proportion total fatty Relative to of C12:0- alcohol of C12:0- alcohol Variant SEQ ID OH, titer, OH, titer, No. NO: 18 30° C. 30° C. 37° C. 37° C. 201 A2F ++ + ++ + 202 A2H ++ + ++ + 203 A2P ++ + 204 A2R + ++ 205 A2S ++ + 206 A2Y ++ + ++ + 207 Q4N ++ ++ ++ + 208 Q4R ++ + 209 Q4S ++ + ++ + 210 Q4W ++ + ++ + 211 Q4Y ++ + ++ + 212 Q5M ++ + + ++ 213 Q5N + ++ ++ + 214 Q6P ++ ++ ++ + 215 Q6R ++ + ++ + 216 Q6S ++ + 217 Q6V ++ + 218 Q6Y ++ + ++ + 219 N8V ++ + ++ + 220 G9V + ++ + ++ 221 S11D + ++ + ++ 222 S11G + ++ + + 223 A12D + + + ++ 224 A12R + ++ + ++ 225 A12T + ++ + ++ 226 S13G + ++ 227 S13L + + + ++ 228 S13V + + + ++ 229 G14L ++ + ++ + 230 G14M ++ ++ ++ + 231 L16G + + + ++ 232 L16I ++ + ++ + 233 L16S + + + ++ 234 E17C + + + ++ 235 E17H + ++ + ++ 236 E17R + ++ + ++ 237 R20K ++ + ++ + 238 E66D + ++ + ++ 239 E66F + + + ++ 240 E66S + ++ + ++ 241 E66Y + + + ++ 242 F68A + + + ++ 243 F68V + + + ++ 244 L69I + ++ + ++ 245 L69M + ++ + ++ 246 L69Q + + + ++ 247 N70D + ++ + ++ 248 N70L + ++ + ++ 249 N70M + ++ + ++ 250 N70R + ++ + ++ 251 N70T + ++ + ++ 252 E71C + + + ++ 253 E71M + + + ++ 254 E71S + + + ++ 255 I72L + ++ + ++ 256 A73G + ++ + ++ 257 A73H + + + ++ 258 A73K + ++ + ++ 259 A73L + ++ + ++ 260 A73M + + + ++ 261 S74L ++ + 262 S74T + ++ + ++ 263 S74W + + + ++ 264 S75C + ++ + ++ 265 S75E + + + ++ 266 S75H + ++ + ++ 267 S75N + ++ + ++ 268 S76E + ++ + ++ 269 S76F + + + ++ 270 S76I + ++ + ++ 271 S76L + + + ++ 272 S76R + + + ++ 273 V77P + + + ++ 274 V77T + ++ + ++ 275 F78M + + + ++ 276 E79D + ++ + ++ 277 E79I + + + ++ 278 E79L + + + ++ 279 E79Q + ++ + ++ 280 E79V + + + ++ 281 R80I + + + ++ 282 R80L + + + ++ 283 L81F + + + ++ 284 L81T + + + ++ 285 A88V + + + ++ 286 F89D ++ + 287 F89N ++ + 288 F89P ++ + 289 F89R ++ + 290 E90D + ++ 291 L93D ++ + 292 E103C + + + ++ 293 E103S + + + ++ 294 E103V + + ++ ++ 295 E106A + + + ++ 296 E106H + ++ + ++ 297 R108E ++ + ++ + 298 P113I + + + ++ 299 H128C + + 300 H128L + + 301 S129D + + 302 A130C + + 303 A130S + + 304 A131P ++ + 305 A131S + + 306 S132H + + 307 V133A + + 308 F135E ++ + 309 K144E ++ + ++ + 310 I145E + + 311 I145H + + 312 L148T ++ ++ + ++ 313 L150P ++ + 314 E151G + + + ++ 315 E151V + + + ++ 316 V153F ++ + 317 A154G + + + ++ 318 A154R ++ + ++ + 319 A155M ++ + ++ + 320 A155R + + + ++ 321 A155W + + ++ ++ 322 S161Y ++ + ++ + 323 N174A ++ + ++ + 324 K176G ++ + ++ + 325 K176I + + + ++ 326 T177D ++ + + + 327 T177E + + + ++ 328 T177L + + ++ ++ 329 T177R ++ ++ + + 330 S178L + ++ ++ + 331 G179S + ++ 332 G179W ++ + 333 Q180C + ++ + ++ 334 I181D + + + + 335 I181E + + + + 336 I181L + ++ + ++ 337 T182G + + + ++ 338 T182I + + ++ ++ 339 T182K + + + ++ 340 T182R + + + ++ 341 V185G + ++ + + 342 V185P + ++ + ++ 343 I186H + ++ + + 344 K187P + ++ + + 345 S188D + ++ + ++ 346 S188E + + ++ ++ 347 S188R + ++ ++ + 348 S188W + ++ + ++ 349 A189L + + + ++ 350 A189N + ++ + ++ 351 G190I + ++ + + 352 G190K ++ ++ + + 353 G190L + ++ + ++ 354 E191V + ++ ++ + 355 E191W ++ + + ++ 356 I193C + ++ + ++ 357 I193L ++ + + ++ 358 R195F + ++ 359 R195H + ++ 360 R195I + ++ 361 R195N ++ ++ 362 R195W + ++ 363 S196D + ++ 364 T197F + ++ 365 D198S ++ ++ 366 L206C ++ ++ 367 V207M + ++ 368 L209Y + ++ 369 Q211H ++ ++ 370 Q211L + ++ 371 Q211N ++ ++ 372 D212F ++ ++ 373 D216G ++ ++ 374 D216Q ++ ++ 375 K218R ++ ++ 376 R220A ++ ++ 377 R220H ++ ++ 378 V225C + ++ 379 V225M + ++ 380 K228H + ++ 381 I235E + ++ 382 N239C + ++ 383 N240Q ++ ++ 384 N240T + ++ 385 Y241F ++ ++ 386 S244R + ++ 387 G264R ++ ++ 388 L267H + ++ 389 T268N + ++ + ++ 390 I275V + ++ 391 S277A + ++ 392 A278C + ++ 393 E280I + ++ 394 E281S + ++ 395 E281Y + ++ 396 P284C + ++ 397 P284Q + ++ 398 W286Y + ++ 399 E288D + + + + 400 E288H + ++ 401 F308I ++ + 402 G310L ++ + 403 S313Q ++ + + + 404 I316L ++ + 405 V318F ++ + ++ + 406 V318L ++ + ++ + 407 V318M ++ + 408 I355F ++ + ++ + 409 I355L ++ + ++ + 410 I355W ++ + 411 I361C ++ + ++ + 412 I361F ++ + ++ + 413 I361L ++ + ++ + 414 D362L + ++ + ++ 415 A373W ++ ++ ++ ++ 416 D376K + ++ + ++ 417 D376P + ++ + ++ 418 D376R + ++ + ++ 419 F387I ++ + 420 F387L ++ + 421 V401T + ++ 422 R487G ++ + 423 L489F ++ + 424 N490C ++ + 425 R491M ++ ++ 426 L494Y + ++ 427 K495C + ++ 428 K495S + ++ 429 E496G + ++ 430 K498G ++ + 431 S501W ++ + 432 S502A ++ + 433 R503C + ++ 434 A504D ++ + 435 A504E + ++ 436 A504S ++ + 437 A505G ++ + 438 A505E ++ + 439 D506L ++ + 440 D506M ++ + 441 D506R ++ + 442 D506W ++ + 443 T507H ++ + 444 D508L + ++ 445 D508M ++ + 446 D509P ++ + 447 D509Q ++ + 448 D509R ++ + 449 K510D + ++ 450 K510E + ++ 451 K510L + ++ 452 K510M ++ + 453 K510N + ++ 454 K510R ++ + 455 K510S + ++ 456 T511D + ++ 457 T511I + ++ 458 T511S + ++ 459 A512M + ++ 460 A512R + ++ + = up to 1.0 fold improvement over FAR variant SEQ ID NO: 18 ++ = >1.0 fold improvement over FAR variant SEQ ID NO: 18

Table 8 provides the relative fold improvement (FIOP) in the proportion of C12:0 fatty alcohols produced and in the increase in total fatty alcohol titer for illustrative variants relative to FAR Variant No. 405 (SEQ ID NO:20) at 30° C. (for Variant Nos. 461-781) or at 37° C. (for Variant Nos. 782-864). In Table 8, the amino acid substitutions listed for each variant correspond to residue positions of SEQ ID NO:20.

TABLE 8 Variant FAR polypeptides and relative fold improvement in proportion of C12:0 fatty alcohols produced and total fatty alcohol titer FIOP in FIOP in total Variant Amino Acid Substitutions Relative to SEQ ID proportion of fatty alcohol No. NO: 20 C12:0-OH titer 461 S178F; I316L; I361F; N490C; S502A ++ + 462 A155T; F308I; I355F; R491M; D506M ++ + 463 A155T; F308I; I355L; L489F; A505G; D506M; T507H ++ + 464 I316L; I361F; N490C; S502A ++ + 465 V15I; I316L; I361F; N490C; S502A ++ + 466 Q4Y; G14L; K144E; A154R; I316L; F387L; D508M ++ + 467 Q6Y; N8V; S74L; R108H; D198S; I355L; A504D ++ + 468 Q6V; R108H; D198S; R220H; I355L; A504D ++ + 469 Q4W; G14L; K144E; S178F; I316L; I361F; N490C; S502A ++ + 470 S178F; A189N; I361F; E496G; K510S ++ + 471 M1G; F308I; I355L; L489F; A505G ++ + 472 S75N; I361F ++ ++ 473 S178F; I316L; I361F; S502A; K510R ++ + 474 V15I; K144E; I316L; F387L; D508M ++ + 475 I361F; N490C; S502A ++ + 476 Q6S; R108E; D198S; R220H; I355L; A504D ++ + 477 I361F; L489F ++ + 478 V15I; F387L; D508M ++ + 479 S178F; I361F ++ + 480 G14L; S178F; I361F ++ + 481 A155T; F308I; I355L; R491M ++ + 482 N8V; S74L; D198S; R220H; I355L; A504S ++ + 483 S178F; A189N; I361F; R410H ++ ++ 484 S75N; E151G; A189N; F308I; I361C ++ + 485 A189N; I361F; K498G; K510E ++ ++ 486 F308I; R491M ++ + 487 A155T; F308I; I355F; D506M; T507H ++ + 488 A155T; F308I; I355F; R491M ++ + 489 Q4W; G14L; K144E; I316L; I361F; N490C ++ + 490 N8V; S74L; R108H; D198S; I355L; A504D ++ + 491 I361F; K498G; K510S ++ + 492 Q4R; G14L; K144E; I166M; I316L; F387L; D508M ++ + 493 M1G; S178F; I316L; I361F; N490C; K498N; S502A ++ + 494 A155T; I361F; E496G; K498G; K510R ++ ++ 495 I275V; F308I; I361C ++ + 496 A155T; F308I; I355L; T507H ++ + 497 S75N; R108H; A189N; I361F; L489F ++ + 498 S75N; A155T; R195W; G310L; I316L; I355F; I361C ++ + 499 A155T; F308I; I355L; L489F; S501W ++ + 500 R108H; R220H; I355L; A504D ++ + 501 S178F; A189N; I361F; Y380H; E496G; K498G; D506M ++ + 502 A155T; F308I; I355F; R491M; S501W ++ + 503 Q6P; S74L; D198S; R220H; I355L; A504D ++ + 504 Q4N; G14L; K144E; G179D; D508M ++ + 505 K144E; I316L; F387L; D508M ++ + 506 S75N; R108H; R195W; V225M; F308I; I361F ++ + 507 A155T; S178F; A189N; I361F; K498G; K510S ++ + 508 S178F; I361F; N490C; S502A ++ + 509 A155T; A189N; I361F; E496G ++ ++ 510 A155T; F308I; I355F; S501W ++ + 511 Q4Y; G14L; G179D; F387L; D508M ++ + 512 A155T; S178F; A189N; I361F; E496G; K510S ++ + 513 R108H; F308I; I361F; L489F ++ + 514 A155T; S178F; I361F; K498G ++ + 515 S178F; A189N; I361F; E496G; K510E ++ + 516 F308I; I355L; L489F ++ + 517 G14L; S178F; I316L; I361F; N490C ++ ++ 518 Q6P; N8E; S74L; D198S; R220H; I355L; A504D ++ + 519 S178F; I316L; I361F; N490C ++ + 520 A155T; S178F; A189N; I361F ++ + 521 S178F; I316L; I361F; N490C; K498G ++ + 522 A155T; A189N; I361F; Y380H; E496G; D506M; K510E ++ ++ 523 S75N; R108H; R195W; I316L; I361F; L489F ++ + 524 A155T; A189N; I361F; E496G; K510E ++ ++ 525 S74L; R108H; D198S; R220H; I355L; A504D ++ + 526 S75N; R108H; A189N; V225M; F308I; I355F ++ + 527 A155T; F308I; I355L ++ + 528 D198S; I355L; A504D ++ + 529 A2G; S75N; A155T; I275V; F308I; I361F ++ + 530 S178F; I361F; K498G ++ ++ 531 A155T; F308I; R491M; A505G ++ + 532 K144E; F387L; D508M ++ + 533 A155T; F308I; I355F; S501W; A505G ++ + 534 A155T; S178F; A189N; I361F; K498G; D506M; K510E ++ + 535 R195W; F308I; I355L; L489F ++ + 536 A155T; S178F; A189N; I361F; E496G; K498G; K510E ++ + 537 S178F; G310L; I361F; N490C; S502A ++ + 538 A155T; F308I; I355L; L489F ++ + 539 S75N; R108H; E151G; I355L; L489F ++ + 540 A155T; S178F; A189N; I361F; E496G; K510E ++ + 541 S75N; R108H; A155T; F308I; I355L; I361C; L489F ++ + 542 G14L; G310L; I361C ++ + 543 I316L; F387L; D508M ++ + 544 Q6P; A504D ++ + 545 I361F; E496G; K510E ++ ++ 546 A155T; S178F; A189N; I361F; E496G; K498G ++ + 547 A155T; S178F; A189N; I361F; E496G ++ + 548 M1G; A155T; F308I; I355L; L489F; T507H ++ + 549 R108H; F387L ++ + 550 Q6P; R108E; S161Y; D198S; R220H; I355L; A504D ++ + 551 Q6C; D47E; S74L; R108H; S161Y; D198S; A504D ++ + 552 S178F; I361F; E496G; K498G ++ ++ 553 F308I; I355W; R491M; D506M ++ + 554 K144E; R220H; I316L; I361C; N490C ++ + 555 R108H; E191V; F387L ++ + 556 R108H; D198S; R220H; I355L ++ + 557 S75N; V225M; F308I; I361L ++ + 558 G14L; K144E; I166M; G179D; F387L ++ + 559 A155T; S178F; A189N; I361F; K498G; K510E ++ + 560 A155T; S178F; A189N; I361F; Y380H; E496G; K498G; K510E ++ + 561 Q6S; S74L; R108H; L148K; D198S; R220H; I355L; A504D ++ + 562 T268N ++ + 563 A155T; F308I; I355W; L489F ++ + 564 N490C; S502A ++ + 565 R108H; E151G; I275V; F308I; I361L ++ + 566 Q4R; G14L; K144E; G179D ++ + 567 G14L; R20K; R220H; G310L; I316L; I361F; K498G ++ + 568 G14L; D47N; I316L; F387L; D508M ++ + 569 K498G; S502A ++ + 570 S75N; R108H; A155T; F308I; I361F ++ + 571 Q6V; R220H; I355W; A504S ++ + 572 F387L ++ + 573 S75N; R108H; A189N; G310L; I355F ++ + 574 Q6C; N8V; D47N; S74L; R108H; R220H ++ ++ 575 Q6C; N8V; S74L; R108H; R220H ++ + 576 S75N; R108H; E151G; A189N; I275V; F308I; I361C ++ ++ 577 M1V; S161Y; S178F; I316L; I361F ++ + 578 S178F; I361F; K510S ++ + 579 G14L; K144E; G179D; I316L; F387L ++ + 580 S178F; G310L; I361F; N490C ++ + 581 K144E; I316L ++ + 582 Q4W; K144E; S178F; I316L; I361C; N490C ++ + 583 K144E; I166M; I316L; F387L ++ + 584 Q6S; S74P; R108Q; L148K; R220H; A504S ++ + 585 G310L; I361F ++ ++ 586 G14L; K144E; S178F; G310L; I361C ++ ++ 587 A155M; S178F; I316L; I361F; N490C; S502A ++ + 588 K144E; D508M ++ + 589 A155T; S178F; A189N; E496G; K498G; K510S ++ + 590 S178F; I316L; I361C; N490C; S502A ++ + 591 K144E; A154R; I316L; F387L ++ + 592 N8V; R108H; R220H; I355W; A504D ++ + 593 S75N; R108H; A189N; I275V; F308I; L489F ++ + 594 R108H; E191V ++ ++ 595 R108H; E151G; R195W; V225M; I275V; I316L; I355F; L489F ++ + 596 K144E; I166M; F387L; D508M ++ + 597 I316L ++ + 598 Q4S; K144E; A154R; I166M; D508M ++ + 599 S178F; I316L; I361F; N490C; K510R ++ + 600 I166M; I316L; D508M ++ + 601 S75N; R108H; E151G; I275V; I361C ++ + 602 G285D; T430A ++ + 603 L148K; D198S; I355W; A504D ++ + 604 R108H; S178F; E191V; ++ ++ 605 V15I; K144E; A154R; I166M; G179D; I316L; F387L; D508M + + 606 R108H + ++ 607 A155T; F308I; I355F; L489F; D506M + + 608 G14L; I166M; I316L; F387L; D508M + + 609 K144E; G179D; I316L; F387L; D508M + + 610 Q6V; N8V; R108E; L148K; S161Y; R220H; I355L; A504D + + 611 Q4N; K144E; G179D; T511P + + 612 S75N; R108H; S178F; A189N; V225M; F308I; I361C + + 613 V15I; A154R; I316L; F387L + ++ 614 S178F; G310L; I316L; I361C; N490C; S502A + + 615 V15I; K144E; G179D; I316L; F387L; D508M + + 616 S75N; R108H; E151G; R195W; G310L; I355L; L489F + + 617 N8V; R108H; D198S; R220H; I355W; A504S + + 618 S75N; R108H; A155T; S178F; R195W; V225M; F308I; I361C + + 619 S75N; K144E; I166M; F387L + + 620 R108H; A154R; E191V + + 621 S178F; A189N; K498G; K510E + + 622 A155T; S178F; A189N + + 623 S74L; R108H; L148K; S161Y; I355L; A504D + + 624 S75N; R108H; E151G; A189N; I275V; F308I; I361L + + 625 R108H; A155T; I316L; I361C; L489F + + 626 K144E; S178F; I316L; I361L; N490C; S502A + + 627 R108H; I166M; F387L + + 628 S75N; R108H; A155T; R195W; V225M; I316L; I361C + + 629 I166M; I316L; F387L; D508M + + 630 A154R; F387L + + 631 E151G; R195W; I275V + + 632 R108Q; S161Y; D198N; I355S; A504D + + 633 R108H; A155M; S178F + + 634 K144E; G179D; D508M + ++ 635 S74L; R108H; L148K; D198S; R220H; I355L; A504S + + 636 K144E; A154R; I166M; D508M + + 637 S178F; I316L; I361C; N490C + + 638 A155T + + 639 S178F; G310V; I361F; N490C + + 640 G14L; G179D; I316L + + 641 S75N; R108H; E151G; A155T; S178F; V225M; I275V + + 642 Q6V; N8V; S74L; R108E; L148K; S161Y; D198S; R220H; I355L; + + A504D 643 I166M; F387L + + 644 G14L; A155M; S161Y; I361C; N490C + + 645 G14L; K144E; S178F; G310L; I361F; S502A + + 646 K144E; A154R; G179D; I316L; F387L; D508M + + 647 M1W; S74L; R108E; L148K; S161Y; D198S; R220H; I355L; + + A504D 648 S75N; R108H; R195W; I361L + + 649 S178F; I316L; I361L; N490C; S502A + + 650 S75N; R108H; A155T; F308I; I355F + + 651 G14L; K144E; S178F; R220H; G310L; N490C + + 652 S74L; R108E; L148K; S161Y; D198S; R220H; I355L; A504D + + 653 S75N; E151G; R195W; V225M; I275V; F308I; I361L + + 654 Q6V; R108E; D198S; I355W; A504D + + 655 R108H; E151G; A189N; I275V; G310L; I361C + ++ 656 G14L; K144E; S178F; G310L; I361C; N490C; S501F; T511S + + 657 A155T; F308I; I355F; L489F + + 658 I355W; A504D + + 659 K144E; I166M; D508M + + 660 S75N; R108H; V225M; I275V; I361L + + 661 Q6P; S74L; L148K; S161Y; D198S; R220H; I355L; A504D + + 662 R108H; A155T; S178F + + 663 S75N; E151G; R195W; G310L; I361F + + 664 G14L; K144E; I166M; G179D; I316L; F387L; D508M + + 665 I166M; E191V + ++ 666 M1V; A155T; F308I; I355F; L489F; T507H + + 667 R220H; I355L; A504D + ++ 668 S75N; R108H; A155T; V225M; I275V; I361C + ++ 669 S75N; R108H; R195W; I275V; G310L + + 670 S178F; I316L; I361L; N490C + + 671 R108H; I166M + + 672 R108H; I275V; I361L + ++ 673 V15I; I166M; I316L; D508M + + 674 Q6P; R108E; L148K; S161Y; D198S; R220H; I355L; A504D + + 675 G14L; K144E; A155M; S178F; R220H; G310L; I361F; K498G; + + S502A 676 Q6Y; R108H; D198S; R220H; I355W; A504D + + 677 S178F; G310V; I316L; I361C; N490C + + 678 Q6V; D198S; R220H; I355W; A504D + + 679 Q4R; G14L; K144E; I166M; G179D; I316L; F387L; D508M + + 680 S178F; G310L; I361C; S502A + + 681 E496G + ++ 682 I166M; S178F + + 683 S75N; R108H; A155T; A189N; V225M; I275V; I361C + + 684 Q4S; A155M; S178F; G310L; S502A + + 685 Q6Y; N8V; S74L; R108E; D198S; R220H; I355W; A504D + + 686 S178F; G310L; I361L; N490C; S502A + + 687 S75N; E151G; R195W; V225M; G310L; I361C; L489F + + 688 S75N; E151G; I275V; I361L + ++ 689 K144E; I166M + ++ 690 M1W; R220H; I355W; A504D + + 691 R491M + + 692 K144E; A154R; G179D; I316L; D508M + + 693 Q4W; S178F; R220H; I316L; I361L + ++ 694 N8V; R108E; S161Y; D198S; R220H; I355W; A504D + + 695 K144E; G179D; I316L + ++ 696 S75N; E151G; S178F; A189N; V225M; I275V; I361C + + 697 N8V; S161Y; R220H; I355W; A504S + + 698 Q6H; N8A; G9C; S74P; R108E; S161Y; R220H; I355W; A504D + + 699 K144E; I166M; I316L + ++ 700 M1R; D198S; I355W; A504D + + 701 G310L; I316L; I361C; N490C; K498G; S502A + + 702 S161Y; D198S; I355W; A504D + + 703 Q6S; R108H; S161Y; D198S; R220H; I355W; A504D + + 704 S178F; G310L; N490C; S502A + + 705 S74L; S161Y; D198S; I355W; A504D + + 706 F308I; I355W; R491M; D506M; T507H + + 707 S161Y; D198S; R220H; I355W; A504D + + 708 K144E; S178F; G310L; I361L; K498G; S502A + + 709 Q6P; V45A; D47E; S74L; S161Y; D198S; I355W; A504D + + 710 S75N; A155T; I275V; F308I + + 711 S74L; R108H; S161Y; I355W; A504D + + 712 G14L; I166M; G179D; F387L; D509G + + 713 Q4W; G14L; K144E; I166M; G179D; I316L; F387L; D508M + + 714 M1G; A2T; S74L; R108H; S161Y; I355W; A504D + + 715 S74L; D198S; R220H; I355W; A504D + + 716 G14L; S178F; G310L; I361F; N490C; S502A + + 717 R108H; S161Y; D198S; R220H; I355W; A504D + + 718 S75N; E151G; G310L; I361L + ++ 719 M1G; S178F; G310L; I361C; N490C; S502A + + 720 M1R; A155T; I355W; S501W + + 721 K144E; I166M; G179D; D508M + + 722 Q6V; S74L; D198S; R220H; I355W; A504D + + 723 Q4Y; G14L; S178F; D212F; G310L; I361C; N490C; S502A + + 724 S75N; E151G; V225M; I275V; G310L; I361F + + 725 S74L; R108H; D198S; R220H; I355W; A504D + + 726 K144E; A154R; G179D; F387L; D508M + ++ 727 G14L; A155W; S178F; R220H; G310L; I361C; N490C; S502A; + + D508M 728 G14L; K144E; S178F; G310L; I361L; N490C + ++ 729 Q4R; G14L; K144E; I166M; I316L; F387L + + 730 Q4R; K144E; A154R; I166M; F387L; D508M + + 731 M1L; S178F; G310L; I361C; N490C; S502A + + 732 S75N; S178F; A189N; L489F + + 733 Q4W; K144E; I316L; F387L; D508M + ++ 734 R108H; G179W; E191V + + 735 K144E; A154R; I166M; I316L; F387L + + 736 G14L; K144E; A154R; I166M; I316L; F387L; D508M + + 737 S75N; E151G; A155T; R195W; V225M; I275V; G310L; I316L; + + I355L; I361C 738 S178F; G310L; I361C; N490C; S502A + + 739 S75N; R108H; R195W; V225M; F308I; G310L; I355L + + 740 G14L; K144E; S178F; G310L; I361F; N490C; K510R + + 741 S75N; I275V; I355F; L489F + + 742 N8V; R108H; S178F; G310L; I361C; N490C; S502A + + 743 S178F; G310L; I361C; N490C; K498G + + 744 R108H; A154R; I166M; E191V; F387L + + 745 Q4S; Q6C; R43H; V45S; D47E; R108H; L148K; S161Y; D198S; + + R220H; I355L; A504D 746 G14L; S178F; G310L; I361L; N490C + + 747 K144E; A155M; S178F; I316L; I361L; N490C + ++ 748 S75N; E151G; V225M; I275V; I316L; I361L; L489F + + 749 S178F; G310L; I361C; N490C + + 750 K144E; A154R; I166M; F387L; D508M + + 751 S178F; G310L; I361L; N490S; K498N; S502A + + 752 S75N; A155T; S178F; R195W; I275V; I361C + + 753 S75N; S178F; A189N; I275V; F308I; I361L + + 754 K144E; I166M; G179D; F387L + + 755 R20K; S178F; D198S; G310L; I361C; N490C; S502A + + 756 R108H; L148K; I166M; F387L + + 757 S75N; A155T; S178F; R195W; G310L; I355L + + 758 S161Y; S178F; G310L; I361C; N490C + + 759 A155T; F308I; I355W; R491M; A505G + + 760 A155T; F308I; I355W; R491M; S501C; T507H + + 761 S75N; E151G; S178F; V225M; F308I; L489F; + + 762 A2R; T3L; Q4N; Q5N; Q6K; A120C; Q138R; D140Y; F308I; + + I355W; A505G 763 G14L; K144E; A154R; I166M; G179D; I316L; F387L; D508M + + 764 S75N; R108H; A155T; S178F; V225M; I275V; F308I + + 765 A155M; G179W; E191V; F387L + + 766 F308I; I355W; S501W + + 767 K144E; I166M; G179D; I316L; F387L; D508M + + 768 G14L; S161Y; S178F; R220H; G310L; I361L; N490C; S502A + + 769 A155T; F308I; I355W + + 770 S178F; G310L; I361L; N490C + + 771 S75N; R108H; A155T; I275V; G310L; I361L + + 772 Q4W; R20K; K144E; S178F; G310L; I361L; N490C; K498G + + 773 K144E; A154R; I166M; G179D; F387L + + 774 K144E; A154R; I166M; G179D; F387L; D508M + + 775 Q4R; K144E; I166M; G179D; I316L; F387L; D508M + + 776 S75N; R108H; F308I; G310L; I361L + + 777 K144E; S178F; D198S; G310L; I361L; N490C + + 778 R108H; I166M; G179W; F387L + + 779 R108H; F387L; V424M + + 780 D85E; R108H; Y247N; S283A; F387L; G417V; N419S; T436A; + + T442I 781 R108H; A154R; A157Q; E158N; L159*; N160T; S178F; E191V; + + F387L 782 T197F; Y221D; R236I; G242E; D245H; G264R; S273F; S283F; ++ + P284L 783 V15I; R195W; V225M; R410H; A504E; K510L ++ + 784 R195W; R410H; A504E ++ ++ 785 V133G; N134D; D140Y ++ ++ 786 V133G ++ + 787 A238G ++ ++ 788 V15I; R195W; V225M; R410H; K510S 1 ++ 789 E280I + ++ 790 Q211R; S215Y; K218P; Y221K; G227E; V231A; S244P; L253V; + ++ L258P 791 E151G; I275V; K495S; E496G; T511I + ++ 792 M1G + ++ 793 S75N; K495S + ++ 794 S75N; I275V; K495S; E496G; T511I + ++ 795 R195W; A504E + ++ 796 Y241F + ++ 797 Q211L; V225C; A278C + ++ 798 V15I; R195W; R410H; A504E + ++ 799 G49E; S188W; E280I + ++ 800 S188W; Y241F + + 801 R195N + ++ 802 S75N; E151G; I275V; K495S; E496G + + 803 R195H + ++ 804 M1E; A2R; T177E; T197F; R220A + ++ 805 T182G; R195N + ++ 806 L267H + ++ 807 T182G; R195I + ++ 808 L209Y; R220A; P284Q + + 809 Q211H + ++ 810 S75N; E151G; K495C; E496G; T511I + ++ 811 V15I; R195W; V225M; R410H; A504E + ++ 812 V15I; R195W; R410H; K510S + ++ 813 V15I; R195W; V225M; R410H + ++ 814 V133A; T182G; R195H; N239C + + 815 S75N; I275V; K495C; T511I + ++ 816 S75N; E496G + ++ 817 V15I; R195W; K510N + ++ 818 S75N; E151G; K495S + ++ 819 V15I; R195W; R410H; K510N + ++ 820 R195N; N239C + + 821 V15I; R195W; V225M; R410H; A504E; K510S + ++ 822 S196D; Q211H; Y241F + + 823 R195W; V225M; K510E + ++ 824 S75N; K495S; T511D + ++ 825 G264R + ++ 826 S75N; I275V; K495C + ++ 827 S75N; I275V; K495S + ++ 828 S75N; E151G; K495C; T511S + ++ 829 S188W; S196D; Y241F + + 830 V15I; R195W; V225M; R410H; K510E + + 831 V15I; R195W; R410H; A504E; K510S + + 832 A164V; I275V; K495S; T511D + + 833 S75N; I275V; E496G + + 834 V15I; R195W; V225M; R410H; A504E; K510D + + 835 V15I; R195W; V225M; R410H; K510D + + 836 V15I; R195W; K510D + + 837 M1L; S188W; Q211H; Y241F + + 838 M1G; V15I; P62Q; R195W; K510D + ++ 839 V15I; R195W; R410H; K510E + ++ 840 T182G; R195N; N239C + + 841 S188W; Q211N; L267H + + 842 S75N; I275V; K495C; T511S + ++ 843 S75N; I275V; K495C; E496D; T511S + ++ 844 V15I; R195W; R410H; K510D + ++ 845 T177E; T197F; R220A; G264R + + 846 S75N; E151G; I275V; E496G + ++ 847 T177E; T197F; G264R; P284Q + + 848 S75N; E151G; I275V; E496G; T511I + ++ 849 S188W; S196D; Q211L; Y241F + + 850 S75N; E151G; I275V; K495C; E496G + ++ 851 Q122H; T182G; R195H; L206C; N239C + + 852 S75N; E151G; I275V; K495C + ++ 853 S75N; E151G; I275V; E496G; T511S + ++ 854 K144R + ++ 855 S75N; E151G; I275V; K495C; T511D + ++ 856 E90Q; T197F; L209Y; R220A; G264R; P284Q + + 857 S75N; E151G; I275V; E496G; T511D + ++ 858 L209Y; P284Q + ++ 859 T197F; R220A; G264R + ++ 860 M1L; S188W; S196D; L267H; E280I + + 861 N134Y; S188W; S196D; Y241F; L267H + + 862 T197F; P284Q + ++ 863 S188W; S196D; Y241F; A278C + + 864 T197F; R220A; P284Q + ++ + = up to 1.0 fold improvement over FAR variant 405 (SEQ ID NO: 20) ++ = >1.0 fold improvement over FAR variant 405 (SEQ ID NO: 20)

Table 9 provides the relative fold improvement (FIOP) in the proportion of C12:0 fatty alcohols produced and in the increase in total fatty alcohol titer for illustrative variants relative to FAR variant 545 (SEQ ID NO:22) at 30° C. In Table 9, the amino acid substitutions listed for each variant correspond to residue positions of SEQ ID NO:22.

TABLE 9 Variant FAR polypeptides and relative fold improvement in proportion of C12:0 fatty alcohols produced and total fatty alcohol titer FIOP in FIOP in total Variant Amino Acid Substitutions Relative to SEQ ID proportion of fatty alcohol No. NO: 22 C12:0-OH titer 865 G14L; A189N; R220H; I316L; I355F; R410H; S502A ++ ++ 866 G14L; I316L; I355F; A504D ++ ++ 867 A189N; R220H; I316L; I355F; R410H; S502A; A504D ++ ++ 868 G14L; R220H; I316L; I355F; S502A ++ ++ 869 G14L; A189N; R220H; I316L; I355 ++ + 870 G14L; A189N; R220H; I316L; I355F; F387L; R410H; S502A ++ + 871 G14L; A189N; R220H; I316L; I355F; F387L; R410H; A504D ++ + 872 I316L; I355F; F387L; R410H; A504D ++ ++ 873 A189N; I316L; I355F; A504D ++ + 874 G14L; A189N; I355F; S502A; A504D ++ ++ 875 I355F; A512M ++ ++ 876 I355F; S502A; A504D ++ ++ 877 G14L; I355F; L489F; S502A; A504D ++ ++ 878 G14L; A189N; R220H; I355F; A504D ++ ++ 879 G14L; A189N; R220H; I355F ++ ++ 880 G14L; R220H; I355F; L489F; S502A; A504D ++ ++ 881 G14L; R220H; I316L; I355F; F387L; R410H; A504D ++ ++ 882 G14L; A189N; I355F; A504D ++ ++ 883 G14L; A189N; R220H; I355F; S502A; A504D ++ ++ 884 I355F ++ ++ 885 Q4H; I355F; D506M ++ ++ 886 A189N; I316L; R410H; E510K ++ ++ 887 I316L; R487G ++ ++ 888 G14L; A189N; I316L; F387L; S502A; A504D ++ ++ 889 I316L; R410H ++ ++ 890 G14L; A189N; I316L; R410H; E510K ++ ++ 891 G14L; I316L; R410H ++ ++ 892 G14L; A189N; I316L; R410H ++ ++ 893 Q6R; R108H; I355L ++ ++ 894 A189N; I316L; R410H ++ ++ 895 Q6V; I355L ++ ++ 896 A2S ++ ++ 897 A2S; I316L ++ ++ 898 Q6V; S74L; R108H; I355L ++ ++ 899 A189N; R220H; I355F; A504D ++ ++ 900 Q6V; R108H; I355L; V401T ++ ++ 901 Q6R; S74L; R108E; L148K; Q167H; I355L; R487G ++ ++ 902 I355L; V401T; R487G ++ ++ 903 I355L; V401T; R487G; A504S ++ ++ 904 K144E; L148K; R491M; D506R; D509Q ++ ++ 905 I316L; R491M ++ ++ 906 K144E; I355L ++ ++ 907 F387L; A512M ++ ++ 908 G14L; T268N; R410H; D508M; E510R ++ ++ 909 A189N; R220H; I355F ++ ++ 910 Q6V; S74L; R108H; K144E; Q167H; I355L ++ ++ 911 K426T ++ ++ 912 I316L; T507H ++ ++ 913 K144E; L148K; R487G; R491M; D506R ++ ++ 914 G14L; A189N; R220H; I355F; F387L; S502A; A504D ++ ++ 915 Q6V; R487G; A504S ++ ++ 916 R487G; R491M ++ ++ 917 G14L; T268N; R410H; E510R ++ ++ 918 Q6R; A154R; I355L; V401T ++ ++ 919 G14L; I316L; E510K ++ ++ 920 G14L; A189N; R220H; I316L; I355F; F387L; R410H; L489F; ++ + S502A; A504D 921 Q6R; A154R; I355L ++ ++ 922 K144E; D509R ++ ++ 923 G14L; A189N; R220H; I355F; F387L; R410H; A504D ++ + 924 G14L; A189N; T268N; R410H; E510K ++ ++ 925 Q6C; R108E; K144E ++ ++ 926 G14L; A189N; I316L; E510K ++ ++ 927 G14L; A189N; R220H; F387L; S502A; A504D ++ ++ 928 K144E; L148K; R491M ++ ++ 929 Q6V; K144E ++ ++ 930 G14L; R220H; F387L; S502A ++ ++ 931 L148K; R491M ++ ++ 932 Q6V; R108H; A504S ++ ++ 933 G14L; A189N; I316L; R410H; D508M; E510R ++ ++ 934 G14L; R410H ++ ++ 935 G14L; R220H; I316L; F387L; R410H; L489F; S502A ++ ++ 936 G14L; I355F; F387L; R410H; L489F; S502A; A504D ++ + 937 R410H; E510K ++ ++ 938 G14L; I316L ++ ++ 939 Q6V; S74L ++ ++ 940 G14L; I355F; L489F; A504D ++ + 941 G14L; A189N; R410H ++ ++ 942 Y500H; A504D; D506M ++ ++ 943 G14L; A189N; R220H; I355F; L489F; A504D ++ + 944 K144E; L148K ++ ++ 945 R491M; D506R ++ ++ 946 G14L; T268N; D508M ++ ++ 947 G14L; T101A; D396G ++ ++ 948 G14L; A189N; R220H; I316L; I355F; F387L; L489F; A504D ++ + 949 I316L ++ ++ 950 T246P ++ ++ 951 G14L; A189N; R220H; L489F; S502A ++ ++ 952 A189N; R220H ++ ++ 953 G14L; R220H; I316L; F387L; L489F; S502A ++ + 954 G14L; T268N; I316L; F387L; S502A ++ + 955 G14L; A189N; E510K ++ ++ 956 A189N; R220H; F387L; A504D ++ ++ 957 G14L; R410H; D508M; E510K ++ ++ 958 G14L; F387L; S502A ++ ++ 959 G14L; T268N ++ ++ 960 G14L; A189N; R220H ++ ++ 961 T507H ++ ++ 962 Q167H ++ ++ 963 R487G; R491M; D509R ++ ++ 964 G14L; A189N; R220H; F387L; L489F; S502A; A504D ++ + 965 G14L; A189N; T268N ++ ++ 966 D509Q ++ ++ 967 G14L; A189N; R220H; T268N; F387L; A504D ++ + 968 E510K ++ ++ 969 R220H; A504D ++ ++ 970 K144E; L148K; S178F; R491M; D506R ++ ++ 971 R108H; L148K; A504S ++ ++ 972 G14L; D508M ++ ++ 973 Q6V; A154R; A504S ++ ++ 974 A189N; R220H; I355F; F387L; L489F ++ + 975 K144E; L148K; R491M; D509Q ++ ++ 976 Q6C; S74L; R108H; L148K; R487G ++ ++ 977 A155T; S178F ++ ++ 978 K144E; L148K; R491M; D506R ++ ++ 979 G14L; R410H; D506M; D508M ++ ++ 980 Q6V; K144E; G179D; I355L; V401T ++ ++ 981 R491M; D506R; D509R ++ ++ 982 G14L; A189N; I316L; F387L; L489F; S502A; A504D; D506G ++ ++ 983 Q6C; K144E; Q167H; G310L; I355L; V401T; R487G ++ ++ 984 G14L; T268N; R410H ++ ++ 985 Q6R; K144E; Q167H; G310L; I355L; R487G; A504S ++ ++ 986 Q6C; Q167H; G310L; I355L; R487G ++ ++ 987 R410H; L489F; S502A ++ ++ 988 Q6C; R136L; Q167H; E205K; G310L; I355L; A504S ++ ++ 989 D506R; D509R ++ ++ 990 Q6V; L148K; L279*; V401T; R487G; A504S ++ + 991 S178F ++ ++ 992 G14L; A189N; T268N; I316L; R410H; E510K ++ + 993 Q6V; K144E; G310L; I355L; V401T ++ ++ 994 R108Q; Q167H; G179D ++ + 995 G14L; A189N; L489F ++ ++ 996 G14L; A189N; T268N; I316L; R410H; E510R ++ + 997 Q6C; G310L; I355L; R487G ++ ++ 998 G14L; A189N; R220H; T268N; A504D ++ + 999 K144E; L148K; D506R; D509Q ++ ++ 1000 L489F; S502A; A504D ++ ++ 1001 Q6R; G310V ++ ++ 1002 S74L; R108E; L148K ++ ++ 1003 Q6C; S74L; R108H; K144E; G310L; I355L; A504S ++ ++ 1004 Q6R; S74L; R108H; K144E; G310L; I355L ++ ++ 1005 G14L; T268N; S502A ++ ++ 1006 G14L; T268N; R410H; D508M ++ ++ 1007 G310L; I355L; V401T ++ ++ 1008 S178F; R491M; D506R; D509Q ++ ++ 1009 G14L; T268N; E510K ++ ++ 1010 Q6V; K144E; G310L; V401T; R487G ++ ++ 1011 A189N; R220H; L489F; S502A; A504D ++ ++ 1012 G14L; A189N; T268N; R410H; D508M; E510R ++ ++ 1013 Q6V; S74L; R108H; Q167H; G310L; A504S ++ ++ 1014 A189N; R220H; I316L; L489F ++ + 1015 Q6V; R108H; G310L; R487G ++ ++ 1016 R220H; L489F; S502A ++ ++ 1017 S178F; R487G; R491M; D509R ++ + 1018 Q6R; R108H; K144E; G310L ++ ++ 1019 G14L; A189N; T268N; D506M; E510K ++ + 1020 A155T; S178F; R491M; D506R; D509Q ++ + 1021 Q6V; G310L; V401T ++ ++ 1022 K144E; L148K; S178F; R491M; D509Q ++ + 1023 Q6C; S74L; G310L; A504S ++ ++ 1024 Q6R; L148K; G310L; I355L; V401T + + 1025 Q6C; S74L; L148K; Q167H; G310L + ++ 1026 Q6R; R108H; L148K; G310L; V401T; A504S + + + = up to 0.5 fold improvement over FAR variant 285 (SEQ ID NO: 22) ++ = >0.5 fold improvement over FAR variant 285 (SEQ ID NO: 22)

Table 10 provides the relative fold improvement (FIOP) in the proportion of C12:0 fatty alcohols produced and in the increase in total fatty alcohol titerfor illustrative variants relative to FAR variant 893 (SEQ ID NO:26) at 30° C. In Table 10, the amino acid substitutions listed for each variant correspond to residue positions of SEQ ID NO:26.

TABLE 10 Variant FAR polypeptides and relative fold improvement in proportion of C12:0 fatty alcohols produced and total fatty alcohol titer FIOP in proportion FIOP in Variant Amino Acid Substitutions Relative to of total fatty No. SEQ ID NO: 26 C12:0-OH alcohol titer 1027 Q4Y; P62Q; A189N; I316L; R410H; ++ + T507H; T511S 1028 P62Q; A189N; I316L; R410H; T511I ++ + 1029 Q4Y; P62Q; I316L; R410H; ++ + T507H; T511I 1030 Q4Y; G14L; P62Q; A189N; I316L; ++ + R410H; T507H 1031 Q4Y; G14L; P62Q; A189N; T197F; ++ + I316L; R410H; T507H 1032 Q4S; P62Q; A189N; I316L; ++ + T507H; T511I 1033 Q4S; G14L; P284L; I316L; T511I ++ + 1034 Q4Y; R410H ++ ++ 1035 Q4Y; G14L; T511S ++ ++ 1036 Q4Y; P62Q; A189N; T507H; T511S ++ ++ 1037 T507H + ++ 1038 T507H; T511I + ++ 1039 Q4Y; P62Q; T507H; T511S + + 1040 Q4Y; A189N + ++ 1041 Q4Y; P62Q; T197F; T511I + ++ 1042 Q4S; A189N; T507H; T511I + ++ 1043 Q4S; P62Q; T507H; T511I + ++ 1044 Q4S; A189N; A504E; T511S + + 1045 Q4S; A189N; T511S + + 1046 Q4S; G14L; P62Q; T511S + + + = up to 1.0 fold improvement over FAR variant 633 (SEQID NO: 26) ++ = >1.0 fold improvement over FAR variant 633 (SEQ ID NO: 26)

Example 7 Chain Length Profile of Fatty Alcohols Exhibited by Representative FAR Variants

The chain length profile of a subset of FAR variants was evaluated as described above in FIG. 2. For each FAR variant evaluated, the total fatty alcohol titer was determined by first adding the titers of each fatty alcohol measured (C10:0-OH, C12:1-OH, C12:0-OH, C13:0-OH, C14:1-OH, C14:0-OH, C15:-OH, C16:1-OH, C16:0-OH, C18:1-OH, and C18:0-OH). The percentage of each fatty alcohol species was then calculated as a percentage of the total fatty alcohols measured. FIG. 1 provides the relative chain length distribution (rounded to the nearest percent) of each of the fatty alcohols C10:0, C12:0, C12:1, C14:0, C14:1, C16:0, C16:1, C18:0, and C18:1 in total fatty alcohol titers produced by recombinant E. coli strains expressing wild-type FAR Maa (SEQ ID NO:2) or a FAR variant having the sequence of SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, or SEQ ID NO:28 when cultured at 30° C.

Example 8 Evaluation of FAR Variants Obtained from Wild-Type M. aquaeolei

Gene acquisition of wild-type M. aquaeolei FAR (“FAR Maq”) is described in the published application WO 2011/008535. The amino acid sequence of M. aquaeolei FAR can be found at GenBank Accession Number YP_(—)959486, and is designated herein as SEQ ID NO:4. The polynucleotide sequence of the codon-optimized gene encoding the FAR polypeptide of SEQ ID NO:4 is designated SEQ ID NO:3. The M. aquaeolei FAR gene and genes encoding variants of the M. aquaeolei FAR were cloned into the vector pCK110900 (depicted as FIG. 3 in published application US 2006/0195947) under the control of a lac promoter as described in WO/2011/008535. The resulting plasmids were introduced into E. coli BW25113 Ltor R (Baba et al., Molecular Systems Biology, 2006 doi:10,1038/msb4100050 Article No. 2006.0008) by routine transformation methods. The engineered E. coli strains comprising a plasmid containing a heterologous gene encoding wild-type and variant FARs were grown and tested as described above in Example 2.

Example 9 Evaluation of M. aquaeolei FAR Variants Using Microtiter Plates

FAR Maq variants were grown in 96-well shallow plates containing 180 μL M9YE medium supplemented with 1% glucose and 30 μg/mL chloramphenicol (CAM), for approximately 16-18 hours (overnight) in a shaker-incubator at 30° C., 200 rpm. A 5% inoculum was used in 96-deep-well plates to initiate fresh 380 μL M9YE medium culture supplemented with 30 μg/mL CAM and 0.5% glucose. The culture was incubated for 2 hours at 30° C., 250 rpm to an OD₆₀₀ of 0.6-0.8, at which point expression of the heterologous FAR gene was induced with isopropyl-β-D-thiogalactoside (IPTG) (1 mM final concentration). Incubation was continued for about 24 hours under the same conditions. An additional amount of glucose (0.5% w/v final conc.) was added to the culture at 6 hours after induction by IPTG. Cell cultures were extracted with 1 mL of isopropanol:methyl t-butyl ether (MTBE) (4:6 ratio) for 2 hours. The extracts were centrifuged and the upper organic phase was transferred into polypropylene 96-well plates and analyzed by the GC-FID method described above in Example 2. Identification of individual fatty alcohol was done by comparison to commercial standards (Sigma Chemical Company, 6050 Spruce St. Louis, Mo. 63103).

Table 11 provides the relative fold improvement (FIOP) in the proportion of C12 and C14 fatty alcohols in the total fatty alcohols produced (i.e., the improvement in the percentage of C12 and C14 fatty alcohols in the total fatty alcohol titer) for illustrative variants relative to wild-type M. aquaeolei FAR at 30° C. Codon-optimized SEQ ID NO:3 was mutated and used to express FAR Maq Variant Nos. 1047-1070. The improvement in proportion of C12 and C14 fatty alcohols was determined as described in Example 2. The improvement in total production of fatty alcohols for variants as compared to SEQ ID NO:4 was in the range of 0.8 to 2.5. In Table 11, the amino acid substitutions listed for each variant correspond to residue positions of SEQ ID NO:4 (e.g., “N135K” means that the residue at position 135 in SEQ ID NO:4 (asparagine) is substituted with lysine), and the amino acid positions were determined by optimal alignment with SEQ ID NO:4.

TABLE 11 Variant FAR polypeptides and relative fold improvement in production of C12 and C14 fatty alcohols. Relative FIOP of Amino acid substitutions C12 and C14 fatty alcohol Variant No. relative to SEQ ID NO: 4 production† 1047 N135K + 1048 A74K + 1049 D430K ++ 1050 E228G + 1051 L503R + 1052 S434K ++ 1053 A512P + 1054 A512S + 1055 A512K ++ 1056 S434F + 1057 A512R ++ 1058 A512G ++ 1059 S434W + 1060 K511G + 1061 A512Q + 1062 A512T + 1063 L503S + 1064 A74L + 1065 H8K + 1066 D411R + 1067 K511P + 1068 D116A + 1069 D116E + 1070 I438V + †+ = 1.0 to 1.5 fold improvement over wild-type M. aquaeolei FAR (SEQ ID NO: 4) ++ = 1.6 to 2.0 fold improvement over wild-type M. aquaeolei FAR (SEQ ID NO: 4). Fatty alcohols measured include: C14:0 (1-tetradecanol), C16:1 (cis Δ⁹-1-hexadecenol), C16:0 (1-hexadecanol), and C18:1 (cis Δ¹¹-1-octadecenol).

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. 

1. A microorganism engineered to produce a fatty alcohol composition, said microorganism comprising a polynucleotide sequence encoding a variant fatty alcohol forming acyl-CoA reductase (FAR) polypeptide, wherein said variant FAR comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:2 and further comprising a substitution at one or more positions selected from positions 18, 65, 128, 134, 138, 177, 188, 224, 226, 405, 418, 433, 458, 487, 502, 508, 509, and 511, wherein the position is numbered with reference to SEQ ID NO:2, and wherein the microorganism produces a fatty alcohol composition comprising at least 20% of C12 and C14 alcohols.
 2. The microorganism of claim 1, wherein the microorganism is a bacterial microorganism.
 3. The microorganism of claim 2, wherein the microorganism is E. coli.
 4. The microorganism of claim 1, wherein the substitution is selected from Q18I/L, R65G, N128C/H/L, N134D/K/R/S/Y, E138Q/R, N177D/E/L/R/T, P188D/E/I/R/S/W, K224R, L226M, P405A/C/F/G/L/V, Q418I/N/R/V, S433H/K/N/R/Y, S458E/N/Q, G487R/Y, L502A/Q/R/S/W, R508D/G/H/L/M, K509D/G/H/P/Q/R, and A511D/G/I/K/P/R/S/T.
 5. The microorganism of claim 4, wherein the substitution is selected from Q18I/L, R65G, N128H, N134S, E138Q, N177T, P188S, K224R, L226M, P405V, Q418V, S433K, S458Q, G487R, L5025, R508D, K509D, and A511T.
 6. The microorganism of claim 1, further comprising a substitution at one or more positions selected from positions 4, 6, 7, 14, 62, 104, 108, 189, 220, 227, 316, 318, 355, 361, 365, 401, 410, 496, 507, and 510, wherein the position is numbered with reference to SEQ ID NO:2.
 7. The microorganism of claim 6, wherein the substitution is selected from Q4/N/R/S/W/Y, Q6C/H/K/P/R/S/V/Y, Q7H/N, G14K/L/M/R/V, P62Q, V104I/M, R108E/G/H/Q, A189L/N, R220A/H, E227G, I316L, V318F/L/M, 1355F/L/S/W, I361C/F/L, M365N, G401A/C/L/S/TN, G410D/H/R, E496D/G, T507H/Q, and K510D/E/L/M/N/R/S.
 8. The microorganism of claim 7, wherein the substitution is selected from Q4Y, Q6R, Q7N, G14L, P62Q, V104I, R108H, A189N, R220H, E227G, 1316L, V318F, I355L, I361F, M365N, G401V, G410H, E496G, T507H, and K510E.
 9. The microorganism of claim 1, comprising the amino acid sequence of any of SEQ ID NOs:6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or
 28. 10. The microorganism of claim 1, comprising substitutions at two or more positions.
 11. The microorganism of claim 1, wherein the microorganism produces a fatty alcohol composition comprising at least 30% of C12 and C14 fatty alcohols.
 12. The microorganism of claim 11, wherein the microorganism produces a fatty alcohol composition comprising at least 60% of C12 and C14 fatty alcohols.
 13. The microorganism of claim 1, wherein the microorganism produces a fatty alcohol composition comprising at least 40% of C12 fatty alcohol.
 14. The microorganism of claim 13, wherein the microorganism produces a fatty alcohol composition comprising from about 40% to about 75% of C12 fatty alcohol.
 15. The microorganism of claim 1, wherein the microorganism produces a fatty alcohol composition comprising no more than about 30% of C14 fatty alcohol.
 16. The microorganism of claim 15, wherein the microorganism produces a fatty alcohol composition comprising from about 20% to about 30% of C14 fatty alcohol.
 17. The fatty alcohol composition produced by the microorganism of claim
 1. 18. A microorganism engineered to produce a fatty alcohol composition, said microorganism comprising a polynucleotide sequence encoding a variant fatty alcohol forming acyl-CoA reductase (FAR) polypeptide, wherein said variant FAR comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:2 or SEQ ID NO:4 and further comprising a substitution at one or more positions selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 23, 40, 43, 44, 45, 47, 49, 50, 52, 61, 62, 63, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 83, 84, 85, 86, 87, 88, 89, 90, 91, 93, 97, 98, 100, 101, 103, 104, 106, 107,
 108. 111, 112, 113, 115, 116, 118, 120, 121, 122, 123, 126, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 140, 144, 145, 148, 150, 151, 153, 154, 155, 156, 157, 158, 160, 161, 162, 163, 164, 166, 167, 174, 176, 177, 178, 179, 180, 181, 182, 185, 186, 187, 188, 189, 190, 191, 192, 193, 195, 196, 197, 198, 200, 205, 206, 207, 208, 209, 211, 212, 215, 216, 218, 220, 221, 224, 225, 226, 227, 228, 231, 235, 236, 238, 239, 240, 241, 242, 244, 245, 246, 247, 253, 258, 263, 264, 266, 267, 268, 270, 273, 275, 277, 278, 280, 281, 283, 284, 285, 286, 288, 303, 306, 308, 310, 313, 316, 318, 331, 337, 338, 339, 341, 351, 352, 355, 359, 361, 362, 363, 365, 368, 370, 373, 374, 376, 377, 380, 382, 384, 387, 388, 389, 393, 396, 397, 398, 399, 400, 401, 402, 404, 405, 406, 308, 409, 410, 411, 412, 413, 414, 416, 417, 418, 419, 421, 424, 426, 429, 430, 432, 433, 436, 442, 443, 446, 452, 458, 463, 465, 466, 474, 478, 482, 487, 489, 490, 491, 494, 495, 496, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, or 512, wherein the position is numbered with reference to SEQ ID NO:2, and wherein the microorganism produces a fatty alcohol composition comprising at least 30% of C12 and C14 fatty alcohols.
 19. The microorganism of claim 18, wherein the substitution is selected from M1E/G/L/R/V/W, A2D/F/G/H/P/Q/R/S/T/W/Y, T3/I/L, Q4/N/R/S/W/Y, Q5M/N, Q6C/H/K/P/R/S/V/Y, Q7H/N, N8A/E/H/V, G9C/F/V, A10T, 511D/G, A12D/R/S/T, S13G/L/V, G14K/L/M/R/V, V15I, L16G/I/S, E17C/G/H/R, Q18I/L, R20K, H23R, K40R, R43H, T44A, V45A/S, D47E/N, G49E, G50A, H52Y, H61R, P62Q, A63D, R65G, E66D/F/S/Y, F68A/V, L69E/I/M/Q, N70D/E/L/M/R/T, E71C/M/Q/S, 172L, A73G/H/K/L/M, S74K/L/P/T/W, S75C/E/H/N, S76E/F/I/L/R, V771/P/T, F78M, E79D/I/L/Q/V, R801/L, L81F/T, H83E, D84A, D85E, N86S, E87V, A88G/V, F89D/N/P/R, E90D/Q, T91A/R, L93D, V97I, H98P, 1100V, T101A, E103C/S/V, V104I/M, E106A/H, S107A, R108E/G/H/Q, L111I, T112G, P113I/Q, R115D, F116Y, A118K, A120C, G121T, Q122E/H/T, V123L, F126V, N128C/H/L, S129D, A130C/S, A131P/S, S132H, V133A/G, N134D/K/R/S/Y, F135E, R136L, E137L, E138Q/R, D140Y, K144A/E/R, 1145E/H, L148E/K/T, L150P, E151G/R/V, V153F/I, A154G/R, A155G/M/R/T/W, L156M, A157Q/V, E158D/N, N160T, S161P/Y, A162K, M163L, A164V, 1166L/M, Q167H, N174A, K176G/I/M, N177D/E/L/R/T, S178F/L, G179D/S/W, Q180C/R, I181D/E/L/V, T182G/I/K/R, V185G/I/P, I186H, K187P, P188D/E/I/R/S/W, A189L/N, G190I/K/L, E191V/W, 5192A, I193C/L/V, R195F/H/I/N/W, S196D, T197F/P, D198S, Y200F, E205K, L206C, V207L/M, H208R, L209N/T/Y, Q211H/L/N/R, D212F, S215E/Y, D216G/Q, K218P/Q/R, R220A/H, Y221D/K, K224R, V225C/M, L226M, E227G, K228H, V231A, 1235E, S236I, A238G, N239C, N240Q/R/T, Y241F, G242E, S244A/P/R, D245H, T246A/P/V, Y247N, L253P/V, L258P, S263N, G264R, S266A/T, L267H, T268N, V270L, 5273F, 1275V, S277A, A278C, E2801, E281S/Y, S283A/E/F/M/T, P284C/L/Q, G285D, W286Y, E288D/H/Q, E303G, S306T/W, F3081, G310LN, 5313Q, I316L, V318F/L/M, S331V, S337G, G338E, S339P, Q341R, G351C, 5352G, I355F/L/S/W, K359E, 1361C/F/L, D362L, Y363H, M365N, A368S, T370A/I, A373W, A374Y, D376K/P/R, Q377H/K, Y380H/R, R382H/Q, T384S, F3871/L, V388L, A3891/M/L/V, K393A, D396G, V397L, V398Y, V3991, G400S, G401A/C/L/S/TN, M402V, V4041/L, P405A/C/F/G/L/V, L406Y, 1408L, A409T/V/W/Y, G410D/H/R, K411R, A412V, M413L/R, R414K, A416L/V, G417V, Q418I/N/R/V, N419S, E421D/G/I/L/N/P/R/S/V, V424M, K426R/T, D429E/K/R, T430A/H/I, R432C/Q, S433H/K/N/R/Y, T436A, T4421, A443T, Y446F, S452E/G/N, S458E/N/Q, L463V, R465K, V466G/Q, Q474L/R, Q478E, C482R, G487R/Y, L489F, N490C/S, R491M, L494Y, K495C/S, E496D/G, K498G/N, L499R/S/V, Y500H/N, S501C/F/W, L502A/Q/R/S/W, R503c/K, A504D/E/S/T, A505E/G/K, D506G/L/M/R/W, T507H/Q, R508D/G/H/L/M, K509D/G/H/P/Q/R, K510D/E/L/M/N/R/S, A511D/G/I/K/P/R/S/T, and/or A512M/R.
 20. The microorganism of claim 18, wherein the variant FAR comprises an amino acid substitution set selected from the substitution sets listed in Table 1, Table 2, Table 4, Table 7, Table 8, Table 9, Table 10, or Table
 11. 21. The microorganism of claim 18, wherein the microorganism is a bacterial microorganism.
 22. The microorganism of claim 21, wherein the microorganism is E. coli.
 23. An isolated variant fatty alcohol forming acyl-CoA reductase (FAR) polypeptide comprising an amino acid substitution set selected from the substitution sets listed in Table 1, Table 2, Table 4, Table 7, Table 8, Table 9, Table 10, or Table
 11. 24. The isolated variant FAR polypeptide of claim 23, comprising the amino acid sequence of any of SEQ ID NOs:6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or
 28. 25. A recombinant polynucleotide comprising a sequence encoding the variant FAR polypeptide of claim
 1. 26. An expression vector comprising the recombinant polynucleotide of claim
 25. 27. A host cell comprising the recombinant polynucleotide of claim
 25. 28. A method of producing a fatty alcohol composition, the method comprising culturing the microorganism of claim 1 in the presence of a carbon source under conditions in which fatty alcohols are produced.
 29. The method of claim 28, further comprising recovering the fatty alcohol composition.
 30. The method of claim 28, wherein at least 2 g/L of recoverable fatty alcohols are produced.
 31. A composition comprising the fatty alcohols, or derivatives thereof, produced by the method of claim
 28. 32. The composition of claim 31, wherein the composition is a detergent composition.
 33. The composition of claim 31, wherein the composition is a personal care composition.
 34. A method of producing a detergent composition, the method comprising: combining the fatty alcohols produced by the method of claim 28, or a fraction thereof, with a detergent component selected from sodium carbonate, a complexation agent, zeolites, a protease, a lipase, amylase, carboxymethyl cellulose, optical brighteners, colorants, and perfumes; thereby producing the detergent composition.
 35. A detergent composition produced by the method of claim
 34. 